CD44v6 increases gastric cancer malignant phenotype by modulating adipose stromal cell-mediated ECM remodeling

Abstract

CD44, an abundantly expressed adhesion molecule, and its alternative splice variants have been associated with tumorigenesis and metastasis. In the context of gastric cancer (GC), de novo expression of CD44 variant 6 (CD44v6) is found in more than 60% of GCs, but its role in the pathogenesis and progression of this type of cancer remains unclear. Using a combination of media conditioning experiments and decellularized extracellular matrices (ECMs), this study investigates the hypothesis that CD44v6 overexpression enhances tumor cell malignant behavior by modulating stromal cell-mediated ECM remodeling. Our findings indicate that soluble factors secreted by CD44v6 expressing GC cells particularly increase proliferation and myofibroblastic differentiation of adipose stromal cells (ASCs). These changes in ASC phenotype mediate the deposition of fibrotic/desmoplastic ECM that, in turn, stimulates GC proliferation and inhibits GC clustering. Pharmacological inhibition of matrix metalloproteinase (MMP) activity in tumor cells abrogated matrix-induced changes in tumor cell malignant behavior. Additionally, studies in mice confirmed the pathological relevance of CD44v6 expression and consequential changes in ECM remodeling to gastric tumorigenesis in vivo. Collectively, these results indicate a direct link between CD44v6, ECM remodeling, and GC malignant behavior opening new insights into potential CD44v6-targeted therapies.

Fig. 1

CD44v6 alters tumor growth and architecture in vivo. (A) Schematic showing the establishment of a gastric cancer (GC) cell line stably expressing CD44v6 and study design for subcutaneous xenograft injections of GC cells individually in SCID mice. (B) Immunofluorescence images showing CD44v6 isoform expression and nuclei (DAPI) on MKN74 and CD44v6 cells. Scale bars = 50 μm. Inset bars = 20 μm. (C) Representative histograms of MKN74 and CD44v6 analysis by flow cytometry quantifying CD44v6 expression. (D) Tumor volume from MKN74 (n = 3) and CD44v6 (n = 8) subcutaneous xenografts over eight weeks of growth and representative gross images of explanted tumors at week 8. Scale bars = 5 mm. (E) Histological grading and representative photomicrographs of the desmoplastic response of MKN74 and CD44v6 subcutaneous xenografts on Hematoxylin and Eosin (H&E) stained sections with confirmation of collagen deposition (blue) via Masson’s Trichrome-stained sections after 8 weeks of subcutaneous injection. Arrows indicate fibrotic tissue. Scale bars = 25 μm, inset bars = 50 μm.

Fig. 2

Tumor-secreted soluble factors from CD44v6 expressing GC cells promote adipose-stromal cell (ASC) myofibroblast differentiation in vitro and desmoplasia in vivo. (A) Experimental setup showing collection of tumor-conditioned media (TCM) from GC cell lines and its incubation with ASCs. (B) Number of ASCs after culture in TCMs collected from Control, MKN74, and CD44v6 cells relative to Control (n = 3 samples per condition). (C) Immunofluorescence analysis of BrdU incorporation in ASCs treated with the different TCMs (n = 40 images per condition). Scale bars = 20 μm. (D) Immunofluorescence analysis of α-smooth muscle actin (α-SMA) of ASCs relative to Control (n = 20 images per condition). Scale bars = 20 μm. (E) Schematic of xenografting conditions. CD44v6 cells were injected into SCID mice either alone (n = 8) or in combination with ASCs (n = 7) and tumors were harvested after 8 weeks. Representative photomicrographs of H&E-stained cross-sections and confirmation of collagen deposition (blue) via Masson’s Trichrome stained sections. Corresponding histological scoring of desmoplasia based on H&E sections. Tumors were harvested 8 weeks after injection. Scale bars = 25 μm, Inset bars = 50 μm. * p < 0.05.

Fig. 3

CD44v6 TCM-treated ASCs increase pro-fibrotic ECM deposition and remodeling. (A) Western blot quantification of fibronectin (Fn) deposited by TCM-treated ASCs relative to the corresponding HSP90 levels (n = 3 samples per condition). (B) Immunofluorescence image analysis of Fn deposited by TCM-treated ASCs relative to Control (n = 20 images per condition). Scale bars = 20 μm. (C) Quantification of deposited ECM thickness by confocal image analysis of Fn (n = 37 areas per condition). Scale bars = 20 μm. (D) Experimental setup illustrating in vitro ECM decellularization. (E and F) Immunofluorescence image analysis comparing Fn content post-decellularization relative to pre-decellularization (F-actin staining of ASCs) across experimental conditions (n = 40 images per condition). Scale bars = 20 μm. FOV, field of view. * p < 0.05.

Fig. 4

Decellularized ECMs assembled by CD44v6-TCM treated ASCs promote GC phenotypes associated with malignancy. (A) Experimental setup to analyze the effect of different ASC-assembled, decellularized ECMs on GC cells (MKN74 and CD44v6 cells). (B) Number of MKN74 and CD44v6 cells after 3 days of culture on decellularized ECMs relative to tissue culture plastic (TCPS) (n = 4) * p < 0.05. (C) BrdU incorporation by MKN74 and CD44v6 cells after 3 days of culture on decellularized ECMs and TCPS determined by immunofluorescence image analysis (n = 20 images per condition) * p < 0.05 from different cell lines within the same experimental condition, # and ◆ p < 0.05 between experimental conditions for the same cell line. (D) Representative immunofluorescence (Fn, F-actin, and nuclei [DAPI]) and phase contrast photomicrographs of MKN74 and CD44v6 cells on decellularized ECMs assembled under different TCM culture conditions. Scale bars = 20 μm. (E) Percentage of individual cells and clusters of MKN74 and CD44v6 cells after 3 days of culture on decellularized ECMs and TCPS as determined by image analysis (n = 25 images per condition) * p < 0.05.

Fig. 5

Decellularized CD44v6-associated ECMs increase GC cell proliferation and decrease cell clustering in an MMP-dependent manner. (A) Representative immunofluorescence images of MKN74 and CD44v6 cells after 3 days of culture on decellularized ECMs assembled under different TCM culture conditions and TCPS in the presence and absence of MMPs inhibitor (batimastat). Scale bars = 20 μm. (B and C) BrdU incorporation by MKN74 cells (B) and CD44v6 cells (C) after culture on decellularized ECMs and TCPS in the presence and absence of batimastat as determined by immunofluorescence image analysis (n = 15 images per condition) * p <0.05 from different culture conditions for the same ECM, # and ◆ p <0.05 between ECMs for the same culture condition. (D) Percentage of individual cells and clusters of MKN74 and CD44v6 cells after culture on decellularized ECMs and TCPS in the presence and absence of batimastat as determined by immunofluorescence image analysis (n = 20 images per condition) * p < 0.05.

Fig. 6

Upon co-injection with ASCs CD44v6 tumors are more proliferative and desmoplastic than MKN74 tumors. (A) Schematic showing the study design for subcutaneous xenograft co-injections of MKN74 and CD44v6 cells with ASCs in SCID mice. (B) Measurement of tumor volume from MKN74 with ASCs (n = 3) and CD44v6 with ASCs (n = 8) subcutaneous xenografts over eight weeks of tumor growth with representative gross images of tumors explanted at the end of the experiment. Scale bars = 5 mm. (C) Comparison of the number of tumors formed per total number of subcutaneous implantations between experimental conditions. Analysis was performed 8 weeks after subcutaneous injection. (D) Representative photomicrographs of H&E stained sections of tumors collected 8 weeks after subcutaneous implantation of MKN74 cells + ASCs and CD44v6 cells + ASCs. Comparison of GC cell proliferation between conditions as quantified by immunohistological analysis of Ki67 positive cells (n = 7). Scale bars = 50 μm. * p < 0.05.