Micropapillary structure: A natural tumor collective invasion model with enhanced stem‐like properties

Abstract

Cancer stem cells aggregate to form clusters, which have enhanced stem‐like properties and metastasis potential. However, the molecular mechanisms underlying the formation of cancer stem cell cluster‐like structures with acquisition of stronger invasion and metastasis abilities remain unclear. Micropapillary carcinoma (MPC) is a subpopulation of small, merulioid, inverted, nonfibrous vascular clusters floating in the stroma present in a range of solid malignant tumors and characterized by frequent vascular/lymphatic vessel invasion and lymph node metastasis. Our results showed that these cell clusters exhibit a stem cell phenotype, supporting the premise that MPC may serve as a promising solid tumor model for studying invasion and metastasis of cancer stem cell clusters. In this review, we discuss the latest advances in MPC research and targeted therapy, focusing on analysis of their stem‐like characteristics, mapping their multiomics characteristics, and elucidating the vascular and immune microenvironment of MPC. The existing MPC organoid model was employed to explore potential breakthroughs in targeted therapy and immunotherapy for cancer stem cell clusters.

INTRODUCTION

Cancer stem cells are a rare subpopulation of cells with the potential of self-renewal and multidirectional differentiation, which are closely related to metastasis, drug resistance, and relapse.¹ Recent studies have shown that cluster-like structures formed by cancer stem cells have a stronger metastasis-driving ability than single cancer stem cell.² Cancer cells aggregate into clusters through homologous interactions of the stem cell marker CD44.³ These CD44⁺ cancer cell clusters display a stronger sphere formation ability, enhanced stem-like characteristics, and significant metastasis.⁴ Furthermore, epidermal growth receptor (EGFR) is reported to promote CD44-mediated cancer cell aggregation. Blockage of EGFR with a specific anti-EGFR monoclonal antibody has been shown to effectively inhibit the formation of cancer stem cell clusters and eliminate lung metastasis mediated by these clusters in triple-negative breast cancer.⁵ Accordingly, decoding the key signal networks and molecular characteristics of cancer stem cell cluster-like structures and their mediation of collective invasion should provide an important breakthrough in overcoming metastasis and providing crucial insights for targeted therapy of refractory tumors. However, solid tumor models for analysis of collective invasion and metastasis of cancer stem cell clusters are lacking at present.

Micropapillary carcinoma (MPC) presents a promising model for investigation of the invasive and metastatic properties of cancer stem cell clusters. MPC is a unique cancer subtype that has attracted much attention due to its specific tissue structure and aggressive phenotype. The micropapillary structure was originally reported in breast cancer by Fisher et al.⁶ in 1980. Subsequently, MPC subtypes were identified in lung,⁷ bladder,⁸ stomach,⁹ and other organs. MPC represents a group of small hollow or morula-like cancer cells without fibrous vascular axis that float in the interstitial space, characterized by the histological feature of “polarity reversal.”¹⁰ MPC maintains a tightly clustered cell-to-cell structure during both vascular/lymphatic vessel invasion and lymph node metastasis (Figure 1). Compared with non-MPC, MPC of the breast, lung, bladder, stomach, colorectum, and salivary gland is characterized by a higher risk of vascular and lymphatic invasion and lymph node metastasis and a poorer prognosis. The specific differences between MPC patients and non-MPC patients are shown in Figure 2. Although the pathogenesis, pathological features, and clinical manifestations of MPC vary in different organs, its highly invasive, and the unique micropapillary structure poses challenges to clinical diagnosis and treatment. We summarized the epidemiology, imaging, clinicopathology, clinical staging, and prognosis of MPC of different solid tumors, as shown in Table 1. In this report, we provide an overview on the molecular basis and evidence of MPC as a reliable solid tumor model for studying cancer stem cell cluster-like structure formation as well as the mechanisms underlying collective invasion and metastasis, with the aim of providing guidance for future drug screening and targeted molecular therapy.

[IMAGE OMITTED. SEE PDF]

[IMAGE OMITTED. SEE PDF]

TABLE 1 Clinical data of micropapillary carcinoma (MPC) in different organs.

	Epidemiology	Imaging characteristics	Clinicopathological characteristics	Clinical staging and prognosis
Breast	Invasive micropapillary carcinoma (IMPC) accounts for about 2%–7% of all invasive breast cancers, while pure IMPC is less common and accounts for 0.9%–2% of breast cancers. The average age is 50–60 years old, mainly women, and only a few men are diagnosed	X-rays showed a dense irregular mass associated with microcalcification. The ultrasound showed an irregular, hypoechoic mass with burrs around the edges. MRI showed a mass with irregular shape, irregular edges, or burr and uneven enhancement, accompanied by non-mass enhancement and axillary lymph node enlargement	Compared with invasive ductal carcinoma (IDC), significant features of IMPC include older age, larger tumor diameter, susceptibility to lymph node metastasis and lymphatic vascular invasion, and increased positive expression of estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER-2)	Compared with IDC, a significant proportion of IMPC patients are classified as grade III on histopathological examination. IMPC is more aggressive, the tumor is larger, the lymph node metastasis rate is higher, and the tumor stage is late
Lung	Micropapillary dominant adenocarcinoma (MPA) is considered to be a high-grade and poorly differentiated subtype with poor prognosis. The incidence of MPA is about 4%–9%	High resolution CT scans showed that MPA is usually a solid nodule but may contain small nonsolid components	Compared with other histological types of lung adenocarcinoma, MPA has a higher rate of lymphatic vessel invasion, visceral pleural invasion, and lymph node metastasis	Even the smallest amount of MPA (micropapillary components less than 5% and greater than 1% of the entire tumor) was associated with a poor overall survival prognosis and an increased risk of recurrence compared with patients with tumors without micropapillary components
Bladder	Micropapillary bladder cancer (MPBC) accounts for 0.7%–2.2% of all urothelial tumors. MPBC predominantly occurs in male individuals, with a male-to-female ratio of 5:1, and the age of onset ranges from 40 to 90 years, with an average age of about 67 years	Computed tomography showed a dense mass lesion with irregular, enhanced mass. MRI showed high-signal-intensity lesions in the bladder	Most MPBC patients have myo-invasive disease with pelvic lymph node metastasis	Micropapillary bladder cancer often appears in late stages (clinical stage T3 or T4). The overall prognosis for MPBC is poor, with 5-year and 10-year survival rates of 54% and 27%, respectively
Stomach	The incidence of gastric MPC is 0.07%, with the highest incidence in the age group of 60–70 years	Gastroscopy showed no difference between MPC and other histological subtypes	The presence of micropapillary components in gastric adenocarcinoma was significantly correlated with invasion depth, lymph node metastasis, lymphatic vascular invasion, and perineural invasion	The overall 5-year survival rate of MPC patients was significantly lower than that of patients with stage I and stage II conventional adenocarcinoma
Colorectum	Colorectal MPC accounts for about 9%–19% of all colon cancers and is most common in men aged 53–72 years	The endoscopic features of MPC are indistinguishable from those of traditional colorectal cancer	The presence of MPC in colorectal cancer is closely related to lymph node metastasis and lymphatic vascular invasion of the tumor	Micropapillary components were strongly associated with poor prognosis. Patients with stage I and II MPC had shorter survival and a prognosis as poor as those with stage III and IV MPC compared with the non-MPC group
Salivary gland	The micropapillary subtype is most common in elderly patients	Computed tomography showed calcification inside the tumor with enhanced margins	All 14 cases of micropapillary salivary duct carcinoma showed positive regional lymph node metastasis, vascular and lymphatic infiltration, and perineural invasion	The micropapillary subtype has a worse clinical prognosis than other subtypes

HIGH STEM CELL-LIKE CHARACTERISTICS OF MPC

Expression of cancer stem cell-related biomarkers

Cancer stem cells can be sorted from various malignant solid tumors via flow cytometry according to different stem-like markers, such as CD44,¹¹ Lgr5,¹² and CXCR4.¹³ In general, the positive subsets of stem cell markers show superior tumorigenic ability compared with negative subsets.¹⁴ However, the mechanisms associated with the expression of these stemness markers in MPC remain to be established. Immunohistochemical (IHC) staining of breast invasive micropapillary carcinoma (IMPC) and invasive ductal carcinoma (IDC) showed that the expression ratio of CD44⁺/CD24⁻ subpopulations in IMPC was significantly higher than that in IDC,¹⁵ indicative of elevated stem-like properties of IMPC. IHC staining of CD44 from 25 pure IMPC cases showed no or weak expression of CD44 in the primary foci of all samples, but re-expression in metastatic tissues of seven of the nine samples with lymph node metastasis was observed.¹⁶ These findings clearly suggest that high stem-like features are conducive to lymph node metastasis of MPC clusters. Other studies have reported that RBMS3 silencing upregulates Lgr5 and other genes by activating the Wnt/β-catenin signaling axis, which contributes to the occurrence and development of lung cancer containing micropapillary components.¹⁷ Furthermore, compared with the control group, breast cancers containing micropapillary components displayed higher protein expression of SDF-1/CXCR4, which was positively correlated with the number of lymph node metastases.¹⁸ The existing data clearly demonstrate that the expression of cancer stem-related markers in MPC is upregulated to some extent (Figure 3A). In view of the organ-specific nature of different cancer stem cell biomarkers, we combined this information with the abnormally high expression of cancer stem cell markers in MPCs and summarized them in Table S1.

[IMAGE OMITTED. SEE PDF]

Activation of stemness-related signaling pathways

Several developmental signaling pathways are involved in stem cell maintenance, including Wnt,¹⁹ Notch,²⁰ Hedgehog,²¹ and Hippo/YAP1.²² Accumulating evidence suggests that development-related signaling pathways are abnormally activated in MPC, reflecting high stem-like characteristics of these subtypes (Figure 3B). For instance, second-generation sequencing of lung adenocarcinomas (LUADs) with and without micropapillary components revealed significant high-frequency mutations in genes of the Wnt signaling pathway in the group with micropapillary components.²³ IHC results of stemness-related molecules revealed significant overexpression of SOX2 and NOTCH3 in colorectal MPC.²⁴ Moreover, the Notch1 signaling pathway is abnormally activated in lymphatic vessels of IMPC of the breast.²⁵ The collective studies clearly demonstrate overactivation of development-related signaling pathways in MPC, which could account for its elevated stem-like characteristics.

Various oncogenic signaling pathways are significantly associated with the maintenance of cancer stemness, including Myc,²⁶ PI3K/Akt/mTOR,²⁷ Stat5,²⁸ and PPAR.²⁹ Abnormal activation of cancer stemness-related oncogenic signaling pathways has been consistently reported in MPC (Figure 3B). For instance, comparison between pure IMPC and IDC cases revealed a high proliferation index and significant amplification of the Myc (8q24) gene in IMPC.³⁰ Plakoglobin has been shown to promote the formation of IMPC cell clusters by activating the PI3K/Akt/Bcl-2 signaling axis.³¹ Second-generation sequencing analysis of micropapillary and nonmicropapillary components of LUAD further showed enrichment of mammalian target of rapamycin (mTOR) mutations in micropapillary components.³² Another study on the potential role of STAT5 in breast cancer showed that IMPC accounted for more than 36% spontaneously formed tumors in transgenic mice with constitutively activated STAT5.³³ Comparison of the RNA expression profiles of bladder MPC and conventional urothelial carcinoma cases showed enrichment of peroxisome proliferator-activated receptor γ (PPARγ).³⁴ The collective findings validate overactivation of multiple stemness-related oncogenic signaling pathways, contributing to the elevated stem-like properties of MPC.

Cancer stemness-associated glucose metabolic reprogramming

Since micropapillary cell clusters lack a fibrous vascular axis and float in empty interstitium, it is unclear how nutrients and energy are taken up to support their strong invasion and metastasis capability. Glucose transporter 1 (GLUT1) is upregulated in clinical tissue samples of colorectal MPC and three-dimensional (3D) spheres simulating micropapillary cell clusters in vitro, with a low proliferation index. Moreover, expression of the glycogen-metabolizing enzymes glycogen synthase (GYS1) and glycogen phosphorylase (PYGL) in the 3D spheres is markedly higher relative to that in adherent HCT116 cells.³⁵ Comparison of 29 MPCs with 32 cases of non-MPC disclosed significantly higher expression of the glucose transporters GLUT1 and GLUT2 in MPC.³⁶ Quantitative analysis of glucose consumption in 112 patients with LUAD showed higher glucose intake in high-grade tumors, including micropapillary subtypes.³⁷ The results suggest that the active glycolytic pathway of MPC potentially serves as the energy source that confers highly invasive and metastatic traits. Notably, abnormal activation of glycolysis is closely related to maintenance of stemness of cancer stem cells.³⁸ For example, CD44v10 preserves the stemness of triple-negative breast cancer and mediates drug resistance by upregulating GLUT1 expression to promote glycolysis.³⁹ The unique metabolic characteristics of MPC are therefore considered crucial in the maintenance of cancer stemness function (Figure 3C).

MULTIOMICS CHARACTERISTICS OF MPC

Genomic characteristics

Full-exome sequencing of LUAD samples revealed higher genomic variations in micropapillary components, including tumor mutation load, intratumoral heterogeneity, and copy number variation. RTK/Ras, Notch, and Wnt signaling pathways are activated in the micropapillary component.⁴⁰ Next-generation sequencing results showed a lower frequency of mononucleotide variation and higher frequency of insertion–deletion mutations in MPC compared with non-MPC LUAD patients. In MPC patients, TP53, CTNNB1 and SMAD4 mutations, and ALK rearrangements/fusions were significantly more frequent. Signaling pathway analyses revealed clear genomic changes in the RTK/RAS/MAPK, cell cycle, Wnt, and PI3K/AKT/mTOR pathways in MPC.²³ Moreover, mutations in genes related to TP53 and cell cycle pathways were more common in high-grade LUAD samples with >20% micropapillary and solid components.⁴¹ The above genomic features suggest increased driver mutation and abnormal activation of cell cycle, energy metabolism, and oncogenic signaling pathways in MPC (Figure 4A).

[IMAGE OMITTED. SEE PDF]

Epigenomic characteristics

Earlier DNA methylation analysis of five histological subtypes of LUAD showed that the micropapillary subtype presents a hypermethylated state associated with shorter recurrence-free and overall survival rates. Functional cluster analysis of 365 genes with hypermethylation provided evidence of gene enrichment mainly in the regulatory pathways of cell morphogenesis.⁴² A preliminary investigation of histone methylation biomarkers in LUAD showed that histone 3 lysine 27 tri-methylation (H3K27me3) is highly expressed in MPC and closely associated with poor prognosis,⁴³ suggestive of epigenetic changes in MPC (Figure 4B).

Transcriptome characteristics

RNA sequencing analysis of LUAD showed that genes with low expression in MPC were significantly enriched in immune and inflammatory responses and cytokine activity, while genes with high expression in MPC were significantly enriched in alpha-amylase activity, extracellular exosomes, and extracellular space.⁴⁴ Differential expression analysis of lncRNAs in 10 paired micropapillary subtypes or solid subtypes of LUAD and neighboring normal tissues showed that 110 lncRNAs were significantly upregulated, while 288 lncRNAs were significantly downregulated. Among these lncRNAs, differential expression of CARD8-AS1 was the most significant.⁴⁵ In addition, a number of bladder cancer-related lncRNAs are downregulated in MPC, including UCA1, LINC00152, and MALAT1.⁴⁶ By full transcriptome RNA sequencing analysis of 23 HGT1 micropapillary bladder cancers and 64 conventional urothelial cancers, we obtained a set of 26 MPC signature genes that are highly enriched in metabolism and metabolic transport, immune response, extracellular matrix, and osteoblast differentiation pathways.⁴⁷ Data from spatial transcriptome studies suggest that IMPC cells have extensive heterogeneity and are closely related to metabolic reprogramming based on lipid metabolism.⁴⁸ Additionally, analysis of small RNA transcriptomes indicates significant differences in expression of let-7b, miR-30c, miR-148a, miR-181a, miR-181a*, and miR-181b between IMPC and IDC.⁴⁹ These transcriptome features suggest that the malignant biological phenotype of MPC is the result of interactions between the internal noncoding RNA-mediated oncogenic driving signals of cancer cell clusters and the external tumor microenvironment consisting of factors involved in inflammation, immunity, extracellular matrix, and metabolism (Figure 4C).

Proteome characteristics

Proteomic analysis of micropapillary- and adhesion-dominant LUADs showed that differential proteins are mainly enriched in tyrosine metabolism, ECM-receptor interaction, extracellular tissue, DNA replication, and cell cycle pathways.⁵⁰ In addition, PPARs, glycan biosynthesis and metabolism, and amino acid metabolism signaling pathways are activated in MPC.⁵¹ Proteomic analysis showed dyshomeostasis of 1331 differentially expressed proteins in IMPC. Phosphorylation proteomic analysis led to the identification of 856 differential phosphorylation sites of 655 proteins. The results of kinase–substrate enrichment analysis showed significant activation of Cyclin-dependent Kinases (CDKs) and p90 RSKs and, conversely, significant inhibition of Protein Kinase A (PKA) and Protein Kinase C (PKC) families in IMPC. Integration of the above three analysis methods further revealed specific activation of the mTORC1/S6K2 signaling pathway in IMPC.⁵² Overall, the proteomic characteristics support significantly enhanced levels of glucose, amino acid, and energy metabolism in MPC (Figure 4D).

POOR PROGNOSIS OF MPC

Vascular/lymphatic vessel invasion

Multiple clinical studies have confirmed increased microvessel density (MVD) in IMPC relative to that in IDC.^53,54 Molecules related to tumor angiogenesis are abnormally elevated in IMPC, including tumor necrosis factor-α (TNF-α) and its receptor TNFR-2⁵⁵ and CD146.⁵³ Tumor transplantation studies in vivo have shown that TNF-α-treated breast cancer stem cells induce intratumoral angiogenesis.⁵⁶ CD146, a protein that interacts directly with VEGFR-2, is required for vascular endothelial growth factor (VEGF) to induce phosphorylation of VEGFR-2, activate the AKT/p38 MAPK/NF-κB pathway, and promote endothelial cell migration and microangiogenesis.⁵⁷

In addition, the lymphatic vessel density (LVD) of IMPC was shown to be significantly higher than that of IDC. VEGF-C and its receptor VEGFR-3 were abnormally elevated in IMPC.⁵⁸ Both Notch1 and Notch4 show protein colocalization with VEGFR-3 in vascular and lymphatic vessels of breast IMPC, and Notch1 is highly activated in the lymphatic endothelium of IMPC. Notch plays a key role in tumor lymphangiogenesis by inducing VEGFR-3 expression at the transcriptional level, improving the endothelial cell response to VEGF-C, and promoting endothelial cell survival.²⁵ In summary, elucidation of the molecular mechanisms promoting tumor vasculature and lymphangiogenesis is expected to provide novel insights that may aid in overcoming invasion and metastasis.

Tumor immune microenvironment

The tumor microenvironment associated with CD1d and PJA2 may be critical for the progression of breast IMPC. IHC results confirmed the lower expression of CD1d and PJA2 in the micropapillary than the IDC region.⁵⁹ CD1d is a factor associated with favorable prognosis of breast cancer.⁶⁰ Downregulation of CD1d is reported to inhibit NKT-mediated antitumor immunity and promote metastasis of breast cancer.⁶¹ However, PAJ2 promotes the polarization of M1 macrophages and transformation of M2 into M1 macrophages.⁶² In IMPC, PJA2 expression is reduced. M2 macrophages may therefore fail to transform into M1 macrophages due to PJA2 deficiency, eventually leading to cancer progression. In addition, a retrospective study on 151 patients diagnosed with gastric cancer after gastrectomy (including five with gastric MPC with an incidence of 3.3%) showed that apoptotic neutrophils could be phagocytosed by MPC cells. This cannibalization of neutrophils by cancer cells may be one of the mechanisms enhancing the growth of gastric MPC.⁶³

Another study on the immunological characteristics of micropapillary subtypes of LUAD showed higher levels of tumor mutation load, T cell infiltration, and immunosuppression in patients with high-proportion MPC tumors compared with those with low-proportion MPC,³² clearly indicating a potential link between micropapillary component content and immunotherapy sensitivity. These results suggest that MPC promotes innate immune escape by forming an immunosuppressive microenvironment. Accordingly, immunotherapy with a combination of anti-PD-1/PD-L1 and anti-CTLA-4 drugs may benefit this group of patients.

ORGANOID CULTURE AND TARGETED THERAPY OF MPC

Intervention research on cluster formation

T47D and MCF7 cells were treated with neutrophil elastase to establish T47D/MCF7 cell clusters simulating IMPC cell clusters in vitro.³¹ Plakoglobin is required for the formation of T47D/MCF7 cell clusters and activates the PI3K/Akt/BCL2 pathway. Inhibition of plakoglobin expression could therefore lead to disaggregation of T47D/MCF7 cell clusters.³¹ A recent study by the group of Liu showed that dynamic hyposialylation of Circulating Tumor Cells (CTCs) or loss of ST6GAL1 facilitates the formation of cluster-like structures with high metastatic potential and renders breast cancer cells in a resting state, allowing escape from paclitaxel-induced toxicity. Initial treatment with a neutralizing antibody of the glycoprotein ST6GAL1 substrate PODXL led to significant inhibition of CTC cluster formation and pulmonary metastasis of triple-negative breast cancer.⁶⁴ Screening results of 2486 FDA-approved compounds showed that Na⁺/K⁺-ATPase inhibitors induced successful dissociation of CTC clusters into single cells by increasing the intracellular Ca²⁺ concentration and inhibiting the formation of cell–cell connections, resulting in DNA methylation remodeling of key stem cell and proliferation-related transcription factor-binding sites.⁶⁵ The collective studies indicate that inhibition of the formation of cancer cell cluster-like structures can significantly suppress metastatic ability and weaken the stem-like characteristics of tumor cells (Figure 5A).

[IMAGE OMITTED. SEE PDF]

Intervention treatments for polarity inversion

The group of Inoue developed a culture method facilitating aggregation of primary colorectal cancer cells into spheres based on the principle of mutual connection and retention among tumor cells, designated cancer tissue-originated spheroids (CTOS).⁶⁶ Using the CTOS model, the researchers showed that upon addition of extracellular matrix components to the cell medium of the suspension culture, the top membrane of CTOS with a nonmicropapillary structure shifted from the outer to lumen surface,⁶⁷ while the top membrane of CTOS with a micropapillary structure shifted toward the outer surface, displaying an inverted polarity state.⁶⁸ Mechanistic analyses revealed higher total protein expression and activation of Ras homology family member A (RhoA) in MPC CTOS relative to non-MPC CTOS. Inhibition of RhoA activity by overexpression of GAP⁶⁹ facilitated complete polarity conversion of MPC-CTOS. In addition, application of Y27632, an active inhibitor of Rho-associated protein kinase (ROCK), a downstream molecule of RhoA, led to complete polarity conversion of MPC-CTOS, both in vitro and in vivo.⁶⁸ Establishment of this MPC-CTOS model thus provides a solid foundation for further research on the mechanisms of polarity inversion of MPC and development of effective therapeutic interventions (Figure 5B).

Studies on a treatment plan based on the molecular characteristics of MPC

A lung cancer cell line, KU-Lu-MPPt3, with micropapillary structure was established using fresh tissue from patients. IHC studies confirmed similar protein expression patterns of KU-Lu-MPPt3 and primary tumor tissue. Cells tested positive for cytokeratin, epithelial cell-adhesion molecule (Ep-CAM), E-cadherin, mucin-1, and TTF-1 under conditions of adhesion, suspension, or 3D culture. Sequencing and fragment analyses of EGFR revealed exon 19 mutations in both KU-Lu-MPPt3 cells and primary lung cancer tissues, specifically, the absence of E746-A750 within the framework. Further experiments using this LUAD cell line with micropapillary components may provide fresh insights that aid in the improvement of chemotherapy, targeted therapy, and immunotherapy strategies⁷⁰ (Figure 5C).

Intervention studies on drug resistance of MPC

Yui et al. reported three methods for inducing MCF7 cells to form 3D spheroids: (1) coculture with neutrophils, (2) adding the soluble fraction of neutrophil lysate, and (3) treatment with neutrophil secretions cathepsin G and neutrophil elastase.⁷¹ IHC analysis confirmed that MUC1 is expressed along the outer surface of the cluster, thus morphologically mimicking the IMPC cell cluster.⁷² Furthermore, MCF7 3D spheroids were less sensitive to doxorubicin than monolayers and displayed high expression of the stemness marker CD44 on the cell membrane. The mechanism underlying hypoxia-inducible factor-1a (HIF-1a) activation in MCF7 3D spheroids involves transcriptional regulation of the MDR-1 gene to increase the expression of P-glycoprotein (Pgp) on the plasma membrane. Treatment of cells with the HIF-1a inhibitor 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole significantly suppressed Pgp expression on the cell surface and increased the accumulation of doxorubicin in MCF7 3D spherical cells.⁷² The collective studies clearly demonstrate that IMPC cell clusters simulated using 3D models are characterized by drug resistance, high stemness, and a low-oxygen environment (Figure 5D).

CONCLUSIONS

Micropapillary carcinoma is characterized by collective stemness, invasion, and metastasis and serves as an independent risk factor for poor prognosis of malignant tumors, such as stage 1 LUAD and breast cancer. Multiomics analysis showed significantly enhanced glucose, lipid, amino acid, and energy metabolism levels of MPC. MPC cell clusters are characterized by “high stemness,” “abnormal metabolism,” and “activation of oncogenic signaling pathway” and closely related to cell adhesion, extracellular matrix, and the immune microenvironment, highlighting their complexity and multifaceted structure. Overall, MPC serves as a promising model for investigating the mechanisms underlying the formation and morphological maintenance of cancer cell clusters with high stem characteristics and exploring the pathways of cell cluster invasion and metastasis, providing novel directions and targets for overcoming tumor progression and therapeutic resistance.

AUTHOR CONTRIBUTIONS

Sisi Li: Conceptualization; writing – original draft. Shuangshu Gao: Data curation. Ling Qin: Data curation; resources. Caixia Ding: Writing – original draft. Jinghui Qu: Data curation; resources. Yifei Cui: Formal analysis. Lixia Qiang: Investigation; methodology. Shengjie Yin: Writing – review and editing. Xiaoyu Zheng: Conceptualization; writing – original draft. Hongxue Meng: Writing – review and editing.

ACKNOWLEDGMENTS

We thank members of our laboratory for discussion and help with preparation of the manuscript.

FUNDING INFORMATION

This research work was supported by the National Natural Science Foundation of China (no. 82103659 to S-SL, no. 82072985 to M-HX, no.82202435 to X-YZ), Heilongjiang Province Innovation Base Award Project (JD2023SJ03), Wu-Jieping Medical Foundation (320.6750.19089-22, 320.6750.19089-48), Beijing Medical Award Foundation (YXJL-2019-1416-0069), Nn10 Project of Harbin Medical University Cancer Hospital (Nn102024-05), Postdoctoral Foundation of Heilongjiang Province (LBH-Z20074), and Harbin Medical University Cancer Hospital Top Young Talent Project (BJQN2021-06). This study was also supported by the Natural Science Foundation of Inner Mongolia Autonomous Region (2022QR08003).

CONFLICT OF INTEREST STATEMENT

No potential conflict of interest was reported by the author(s). None of the authors of this manuscript is a current editor or editorial board member of Cancer Science. All the figures were created with .

ETHICS STATEMENT

Approval of the research protocol by an institutional reviewer board: N/A.

Informed consent: N/A.

Registry and the registration No. of the study/trial: N/A.

Animal studies: N/A.

References

Clara JA, Monge C, Yang Y, Takebe N. Targeting signalling pathways and the immune microenvironment of cancer stem cells—a clinical update. Nat Rev Clin Oncol. 2020;17(4):204‐232.

Kapeleris J, Zou H, Qi Y, et al. Cancer stemness contributes to cluster formation of colon cancer cells and high metastatic potentials. Clin Exp Pharmacol Physiol. 2020;47(5):838‐847.

Kawaguchi M, Dashzeveg N, Cao Y, et al. Extracellular domains I and II of cell‐surface glycoprotein CD44 mediate its trans‐homophilic dimerization and tumor cluster aggregation. J Biol Chem. 2020;295(9):2640‐2649.

Liu X, Taftaf R, Kawaguchi M, et al. Homophilic CD44 interactions mediate tumor cell aggregation and polyclonal metastasis in patient‐derived breast cancer models. Cancer Discov. 2019;9(1):96‐113.

Liu X, Adorno‐Cruz V, Chang YF, et al. EGFR inhibition blocks cancer stem cell clustering and lung metastasis of triple negative breast cancer. Theranostics. 2021;11(13):6632‐6643.

Fisher ER, Palekar AS, Redmond C, Barton B, Fisher B. Pathologic findings from the National Surgical Adjuvant Breast Project (protocol no. 4). VI. Invasive papillary cancer. Am J Clin Pathol. 1980;73(3):313‐322.

Emoto K, Eguchi T, Tan KS, et al. Expansion of the concept of micropapillary adenocarcinoma to include a newly recognized filigree pattern as well as the classical pattern based on 1468 stage I lung adenocarcinomas. J Thorac Oncol. 2019;14(11):1948‐1961.

Abufaraj M, Foerster B, Schernhammer E, et al. Micropapillary urothelial carcinoma of the bladder: a systematic review and meta‐analysis of disease characteristics and treatment outcomes. Eur Urol. 2019;75(4):649‐658.

Zhang Q, Ming J, Zhang S, Li B, Yin L, Qiu X. Micropapillary component in gastric adenocarcinoma: an aggressive variant associated with poor prognosis. Gastric Cancer. 2015;18(1):93‐99.

Onuma K, Inoue M. Abnormality of apico‐basal polarity in adenocarcinoma. Cancer Sci. 2022;113(11):3657‐3663.

Takaishi S, Okumura T, Tu S, et al. Identification of gastric cancer stem cells using the cell surface marker CD44. Stem Cells. 2009;27(5):1006‐1020.

Shimokawa M, Ohta Y, Nishikori S, et al. Visualization and targeting of LGR5(+) human colon cancer stem cells. Nature. 2017;545(7653):187‐192.

Cheng CW, Liao WL, Chen PM, et al. MiR‐139 modulates cancer stem cell function of human breast cancer through targeting CXCR4. Cancers (Basel). 2021;13(11): [eLocator: 2582].

Han J, Won M, Kim JH, et al. Cancer stem cell‐targeted bio‐imaging and chemotherapeutic perspective. Chem Soc Rev. 2020;49(22):7856‐7878.

Li W, Liu F, Lei T, et al. The clinicopathological significance of CD44⁺/CD24⁻/low and CD24⁺ tumor cells in invasive micropapillary carcinoma of the breast. Pathol Res Pract. 2010;206(12):828‐834.

Badyal RK, Bal A, das A, Singh G. Invasive micropapillary carcinoma of the breast: Immunophenotypic analysis and role of cell adhesion molecules (CD44 and E‐cadherin) in nodal metastasis. Appl Immunohistochem Mol Morphol. 2016;24(3):151‐158.

Vaishnavi A, Juan J, Jacob M, et al. Transposon mutagenesis reveals RBMS3 silencing as a promoter of malignant progression of BRAFV600E‐driven lung tumorigenesis. Cancer Res. 2022;82(22):4261‐4273.

Liu F, Lang R, Wei J, et al. Increased expression of SDF‐1/CXCR4 is associated with lymph node metastasis of invasive micropapillary carcinoma of the breast. Histopathology. 2009;54(6):741‐750.

Zhang Y, Wang X. Targeting the Wnt/β‐catenin signaling pathway in cancer. J Hematol Oncol. 2020;13(1):165.

Fendler A, Bauer D, Busch J, et al. Inhibiting WNT and NOTCH in renal cancer stem cells and the implications for human patients. Nat Commun. 2020;11(1):929.

Zhu R, Gires O, Zhu L, et al. TSPAN8 promotes cancer cell stemness via activation of sonic hedgehog signaling. Nat Commun. 2019;10(1):2863.

Lu T, Li Z, Yang Y, et al. The hippo/YAP1 pathway interacts with FGFR1 signaling to maintain stemness in lung cancer. Cancer Lett. 2018;423:36‐46.

Li P, Liu L, Wang D, et al. Genomic and clinicopathological features of lung adenocarcinomas with micropapillary component. Front Oncol. 2022;12: [eLocator: 989349].

Lee HJ, Eom DW, Kang GH, et al. Colorectal micropapillary carcinomas are associated with poor prognosis and enriched in markers of stem cells. Mod Pathol. 2013;26(8):1123‐1131.

Shawber CJ, Funahashi Y, Francisco E, et al. Notch alters VEGF responsiveness in human and murine endothelial cells by direct regulation of VEGFR‐3 expression. J Clin Invest. 2007;117(11):3369‐3382.

Moumen M, Chiche A, Decraene C, et al. Myc is required for β‐catenin‐mediated mammary stem cell amplification and tumorigenesis. Mol Cancer. 2013;12(1):132.

Xia P, Xu XY. PI3K/Akt/mTOR signaling pathway in cancer stem cells: from basic research to clinical application. Am J Cancer Res. 2015;5(5):1602‐1609.

Subramaniam D, Angulo P, Ponnurangam S, et al. Suppressing STAT5 signaling affects osteosarcoma growth and stemness. Cell Death Dis. 2020;11(2):149.

Yang L, Shi P, Zhao G, et al. Targeting cancer stem cell pathways for cancer therapy. Signal Transduct Target Ther. 2020;5(1):8.

Marchiò C, Iravani M, Natrajan R, et al. Genomic and immunophenotypical characterization of pure micropapillary carcinomas of the breast. J Pathol. 2008;215(4):398‐410.

Huang L, Ji H, Yin L, et al. High expression of Plakoglobin promotes metastasis in invasive micropapillary carcinoma of the breast via tumor cluster formation. J Cancer. 2019;10(12):2800‐2810.

Zhang S, Xu Y, Zhao P, et al. Integrated analysis of genomic and immunological features in lung adenocarcinoma with micropapillary component. Front Oncol. 2021;11: [eLocator: 652193].

Iavnilovitch E, Cardiff RD, Groner B, Barash I. Deregulation of Stat5 expression and activation causes mammary tumors in transgenic mice. Int J Cancer. 2004;112(4):607‐619.

Guo CC, Dadhania V, Zhang L, et al. Gene expression profile of the clinically aggressive micropapillary variant of bladder cancer. Eur Urol. 2016;70(4):611‐620.

Vyas M, Patel N, Celli R, Wajapeyee N, Jain D, Zhang X. Glucose metabolic reprogramming and cell proliferation arrest in colorectal micropapillary carcinoma. Gastroenterology Res. 2019;12(3):128‐134.

Nosaka K, Makishima K, Sakabe T, et al. Upregulation of glucose and amino acid transporters in micropapillary carcinoma. Histol Histopathol. 2019;34(9):1009‐1014.

Suárez‐Piñera M, Belda‐Sanchis J, Taus A, et al. FDG PET‐CT SUVmax and IASLC/ATS/ERS histologic classification: a new profile of lung adenocarcinoma with prognostic value. Am J Nucl Med Mol Imaging. 2018;8(2):100‐109.

Deshmukh A, Deshpande K, Arfuso F, Newsholme P, Dharmarajan A. Cancer stem cell metabolism: a potential target for cancer therapy. Mol Cancer. 2016;15(1):69.

Guo Q, Qiu Y, Liu Y, et al. Cell adhesion molecule CD44v10 promotes stem‐like properties in triple‐negative breast cancer cells via glucose transporter GLUT1‐mediated glycolysis. J Biol Chem. 2022;298(11): [eLocator: 102588].

Meng F, Zhang Y, Wang S, et al. Whole‐exome sequencing reveals the genomic features of the micropapillary component in ground‐glass opacities. Cancers (Basel). 2022;14(17): [eLocator: 4165].

Ahn B, Yoon S, Kim D, et al. Clinicopathologic and genomic features of high‐grade pattern and their subclasses in lung adenocarcinoma. Lung Cancer. 2022;170:176‐184.

Ito Y, Usui G, Seki M, et al. Association of frequent hypermethylation with high grade histological subtype in lung adenocarcinoma. Cancer Sci. 2023;114(7):3003‐3013.

Zhang Y, Zheng B, Lou K, Xu X, Xu Y. Methylation patterns of Lys9 and Lys27 on histone H3 correlate with patient outcome and tumor progression in lung cancer. Ann Diagn Pathol. 2022;61: [eLocator: 152045].

Sata Y, Nakajima T, Fukuyo M, et al. High expression of CXCL14 is a biomarker of lung adenocarcinoma with micropapillary pattern. Cancer Sci. 2020;111(7):2588‐2597.

Pan C, Wang Q, Wang H, Deng X, Chen L, Li Z. LncRNA CARD8‐AS1 suppresses lung adenocarcinoma progression by enhancing TRIM25‐mediated ubiquitination of TXNRD1. Carcinogenesis. 2023;45:311‐323.

de Jong JJ, Valderrama BP, Perera J, et al. Non‐muscle‐invasive micropapillary bladder cancer has a distinct lncRNA profile associated with unfavorable prognosis. Br J Cancer. 2022;127(2):313‐320.

Bowden M, Nadal R, Zhou CW, et al. Transcriptomic analysis of micropapillary high grade T1 urothelial bladder cancer. Sci Rep. 2020;10(1):20135.

Lv J, Shi Q, Han Y, et al. Spatial transcriptomics reveals gene expression characteristics in invasive micropapillary carcinoma of the breast. Cell Death Dis. 2021;12(12):1095.

Li S, Yang C, Zhai L, et al. Deep sequencing reveals small RNA characterization of invasive micropapillary carcinomas of the breast. Breast Cancer Res Treat. 2012;136(1):77‐87.

Zhou J, Liu B, Li Z, et al. Proteomic analyses identify differentially expressed proteins and pathways between low‐risk and high‐risk subtypes of early‐stage lung adenocarcinoma and their prognostic impacts. Mol Cell Proteomics. 2021;20: [eLocator: 100015].

Xu L, Su H, Zhao S, et al. Development of the semi‐dry dot‐blot method for intraoperative detecting micropapillary component in lung adenocarcinoma based on proteomics analysis. Br J Cancer. 2023;128(11):2116‐2125.

Chen X, Lin Y, Jin X, et al. Integrative proteomic and phosphoproteomic profiling of invasive micropapillary breast carcinoma. J Proteome. 2022;257: [eLocator: 104511].

Li W, Yang D, Wang S, et al. Increased expression of CD146 and microvessel density (MVD) in invasive micropapillary carcinoma of the breast: comparative study with invasive ductal carcinoma‐not otherwise specified. Pathol Res Pract. 2011;207(12):739‐746.

Cui LF et al. Significance of interleukin‐1beta expression and microvascular density in invasive micropapillary carcinoma of breast. Zhonghua Bing Li Xue Za Zhi. 2008;37(9):599‐603.

Cui LF, Guo XJ, Wei J, et al. Overexpression of TNF‐alpha and TNFRII in invasive micropapillary carcinoma of the breast: clinicopathological correlations. Histopathology. 2008;53(4):381‐388.

Narasimhan H, Ferraro F, Bleilevens A, Weiskirchen R, Stickeler E, Maurer J. Tumor necrosis factor‐α (TNFα) stimulate triple‐negative breast cancer stem cells to promote Intratumoral invasion and Neovasculogenesis in the liver of a xenograft model. Biology (Basel). 2022;11(10): [eLocator: 1481].

Jiang T, Zhuang J, Duan H, et al. CD146 is a coreceptor for VEGFR‐2 in tumor angiogenesis. Blood. 2012;120(11):2330‐2339.

Li YS, Kaneko M, Amatya VJ, Takeshima Y, Arihiro K, Inai K. Expression of vascular endothelial growth factor‐C and its receptor in invasive micropapillary carcinoma of the breast. Pathol Int. 2006;56(5):256‐261.

Kanomata N, Kurebayashi J, Koike Y, Yamaguchi R, Moriya T. CD1d‐ and PJA2‐related immune microenvironment differs between invasive breast carcinomas with and without a micropapillary feature. BMC Cancer. 2019;19(1):76.

Canchis PW, Bhan AK, Landau SB, Yang L, Balk SP, Blumberg RS. Tissue distribution of the non‐polymorphic major histocompatibility complex class I‐like molecule, CD1d. Immunology. 1993;80(4):561‐565.

Hix LM, Shi YH, Brutkiewicz RR, Stein PL, Wang CR, Zhang M. CD1d‐expressing breast cancer cells modulate NKT cell‐mediated antitumor immunity in a murine model of breast cancer metastasis. PLoS One. 2011;6(6): [eLocator: e20702].

Zhong J, Wang H, Chen W, et al. Ubiquitylation of MFHAS1 by the ubiquitin ligase praja2 promotes M1 macrophage polarization by activating JNK and p38 pathways. Cell Death Dis. 2017;8(5): [eLocator: e2763].

Barresi V, Branca G, Ieni A, Rigoli L, Tuccari G, et al. Phagocytosis (cannibalism) of apoptotic neutrophils by tumor cells in gastric micropapillary carcinomas. World J Gastroenterol. 2015;21(18):5548‐5554.

Dashzeveg NK, Jia Y, Zhang Y, et al. Dynamic glycoprotein Hyposialylation promotes chemotherapy evasion and metastatic seeding of quiescent circulating tumor cell clusters in breast cancer. Cancer Discov. 2023;13(9):2050‐2071.

Gkountela S, Castro‐Giner F, Szczerba BM, et al. Circulating tumor cell clustering shapes DNA methylation to enable metastasis seeding. Cell. 2019;176(1–2):98‐112.e14.

Kondo J, Endo H, Okuyama H, et al. Retaining cell‐cell contact enables preparation and culture of spheroids composed of pure primary cancer cells from colorectal cancer. Proc Natl Acad Sci USA. 2011;108(15):6235‐6240.

Okuyama H, Kondo J, Sato Y, et al. Dynamic change of polarity in primary cultured spheroids of human colorectal adenocarcinoma and its role in metastasis. Am J Pathol. 2016;186(4):899‐911.

Onuma K, Sato Y, Okuyama H, et al. Aberrant activation of rho/ROCK signaling in impaired polarity switching of colorectal micropapillary carcinoma. J Pathol. 2021;255(1):84‐94.

Kusama T, Mukai M, Endo H, et al. Inactivation of rho GTPases by p190 RhoGAP reduces human pancreatic cancer cell invasion and metastasis. Cancer Sci. 2006;97(9):848‐853.

Matsuo Y, Shiomi K, Sonoda D, et al. Molecular alterations in a new cell line (KU‐Lu‐MPPt3) established from a human lung adenocarcinoma with a micropapillary pattern. J Cancer Res Clin Oncol. 2018;144(1):75‐87.

Yui S, Tomita K, Kudo T, Ando S, Yamazaki M. Induction of multicellular 3‐D spheroids of MCF‐7 breast carcinoma cells by neutrophil‐derived cathepsin G and elastase. Cancer Sci. 2005;96(9):560‐570.

Doublier S, Belisario DC, Polimeni M, et al. HIF‐1 activation induces doxorubicin resistance in MCF7 3‐D spheroids via P‐glycoprotein expression: a potential model of the chemo‐resistance of invasive micropapillary carcinoma of the breast. BMC Cancer. 2012;12:4.