Cancer Cancer-associated Fibroblasts Research Articles

Cancer-associated fibroblast cell surface markers as potential biomarkers or therapeutic targets in lung cancer

Cancer-associated fibroblasts (CAFs) are the vital constituent of the tumor microenvironment, and in communication with other cells, they contribute to tumor progression and metastasis. Fibroblasts are the proposed origin of CAFs, which are mediated by pro-inflammatory cytokines and the recruitment of immune cells akin to wound healing. Although various studies have identified different subpopulations of CAFs in lung cancer, the heterogeneity of CAFs, particularly in lung cancer, and their potential as a therapeutic target remain largely unknown. Notwithstanding CAFs were previously thought to have predominantly tumor-promoting features, their pro- or anti-tumorigenic properties may depend on various conditions and cell origins. The absence of distinct markers to identify CAF subpopulations presents obstacles to the successful therapeutic targeting and treatment of CAFs in cancer. Human clinical and animal studies targeting CAFs have shown that targeting CAFs exacerbates the disease progression, suggesting that subpopulations of CAFs may exert opposing functions in cancer progression. Therefore, it is essential to pinpoint specific markers capable of characterizing these subpopulations and revealing their mechanisms of function. The cell-specific surface markers of CAFs will serve as an initial step in investigating precise CAF subpopulations and their role in diagnosing and targeting therapy against cancer-promoting CAF subsets in lung cancer.

Abstract 2941: A novel gene signature predicting fibroblast composition in the TME allows for an improved selection of patient-derived xenograft (PDX) models for preclinical studies

Abstract The Tumor MicroEnvironment (TME) plays a pivotal role in driving tumor progression, metastasis, and therapeutic resistance through diverse mechanisms. Notably, the TME influences the response to therapy by regulating immune surveillance evasion in tumor cells. Fibroblasts can play a significant role in resistance to therapy. In the context of cancer cancer-associated fibroblasts (CAFs) in the TME have been associated with resistance to various therapeutic approaches. CAFs can contribute to therapy resistance through multiple mechanisms, including extracellular matrix remodeling, immune modulation, angiogenesis and paracrine signaling. Understanding the intricate interactions between fibroblasts and cancer cells is crucial for developing more effective therapeutic strategies and overcoming resistance in cancer treatment. In the pre-clinical stages of therapy testing, the use of clinically and biologically relevant models is essential. However, current preclinical tumor models often lack a comprehensive characterization of the TME. To address this limitation, Champions Oncology utilized transcriptomic data from its exclusive TumorGraft models bank to develop a signature predicting TME composition. Validation involved confirming the predicted TME composition through histological analysis of Patient-Derived Xenograft (PDX) models grown in humanized mice. In an immune-competent host, the Tumor Microenvironment (TME) of the model more accurately replicates the conditions observed in cancer patients. PDX) models are recognized as accurate and clinically relevant for pre-clinical studies. Consequently, Champions Oncology built its pre-clinical research on a deeply characterized PDX bank, employing multi-omic datasets, including WES,RNAseq, proteomics, phospho-proteomics, kinase activity, and patient treatment history. To identify molecular signatures predictive of fibroblast presence, Champions analyzed the data with xCell3 computational method. Concurrently, mice were humanized, and high and low infiltration predicted PDXs models were implanted. Tissues were collected and stained for fibroblast populations. Immunohistochemistry analyses confirmed a strong correlation between the infiltration predicted by the molecular signature and positive staining. The application of this method to Champions' model bank provided a comprehensive understanding of the TME profile, culminating in the definition of a gene signature predictive of fibroblast infiltration. Histological analysis validated the computational findings, creating a TME atlas of the PDX bank. This data support the use of the pre-defined molecular signature, facilitating the pairing of ideal model systems with appropriate humanized hosts for future preclinical studies. Citation Format: Mara Gilardi, Gilad Silberberg, Hsiu-Wen Tsai, Stefano Cairo, Clare Killick-Cole, Michael Ritchie. A novel gene signature predicting fibroblast composition in the TME allows for an improved selection of patient-derived xenograft (PDX) models for preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2941.

Epithelial cells activate fibroblasts to promote esophageal cancer development.

Esophageal squamous-cell carcinoma (ESCC) develops through multistage epithelial cancer formation, i.e., from normal epithelium, low- and high-grade intraepithelial neoplasia to invasive carcinoma. However, how the precancerous lesions progress to carcinoma remains elusive. Here, we report a comprehensive single-cell RNA sequencing and spatial transcriptomic study of 79 multistage esophageal lesions from 29 patients with ESCC. We reveal a gradual and significant loss of ANXA1 expression in epithelial cells due to its transcription factor KLF4 suppression along the lesion progression. We demonstrate that ANXA1 is a ligand to formyl peptide receptor type 2 (FPR2) on fibroblasts that maintain fibroblast homeostasis. Loss of ANXA1 leads to uncontrolled transformation of normal fibroblasts into cancer-associated fibroblasts (CAFs), which can be enhanced by secreted TGF-β from malignant epithelial cells. Given the role of CAFs in cancer, our study underscores ANXA1/FPR2 signaling as an important crosstalk mechanism between epithelial cells and fibroblasts in promoting ESCC.

The advent of immune stimulating CAFs in cancer.

The theory that cancer-associated fibroblasts (CAFs) are immunosuppressive cells has prevailed throughout the past decade. However, recent high-throughput, high-resolution mesenchyme-directed single-cell studies have harnessed computational advances to functionally characterize cell states, highlighting the existence of immunostimulatory CAFs. Our group and others have uncovered and experimentally substantiated key functions of cancer antigen-presenting CAFs in T cell immunity, both in vitro and in vivo, refuting the conventional assumption that CAFs impede adaptive immune rejection of tumours. In this Perspective, I unify the follicular and non-follicular, non-endothelial stroma of tumours under the 'peripheral adaptive immune mesenchyme' framework and position subsets of CAFs as direct positive regulators of the adaptive immune system. Building on the understanding of cancer antigen presentation by CAFs and the second touch hypothesis, which postulates that full T cell polarization requires interaction with antigen-presenting cells in the non-lymphoid tissue where the antigen resides, I re-design the 'cancer-immunity cycle' to incorporate intratumoural activation of cancer-specific CD4+ T cells. Lastly, a road map to therapeutic harnessing of immunostimulatory CAF states is proposed.

Secretome of Stromal Cancer-Associated Fibroblasts (CAFs): Relevance in Cancer.

The cancer secretome reflects the assortment of proteins released by cancer cells. Investigating cell secretomes not only provides a deeper knowledge of the healthy and transformed state but also helps in the discovery of novel biomarkers. Secretomes of cancer cells have been studied in the past, however, the secretome contribution of stromal cells needs to be studied. Cancer-associated fibroblasts (CAFs) are one of the predominantly present cell populations in the tumor microenvironment (TME). CAFs play key role in functions associated with matrix deposition and remodeling, reciprocal exchange of nutrients, and molecular interactions and signaling with neighboring cells in the TME. Investigating CAFs secretomes or CAFs-secreted factors would help in identifying novel CAF-specific biomarkers, unique druggable targets, and an improved understanding for personalized cancer diagnosis and prognosis. In this review, we have tried to include all studies available in PubMed with the keywords "CAFs Secretome". We aim to provide a comprehensive summary of the studies investigating role of the CAF secretome on cancer development, progression, and therapeutic outcome. However, challenges associated with this process have also been addressed in the later sections. We have highlighted the functions and clinical relevance of secretome analysis in stromal CAF-rich cancer types. This review specifically discusses the secretome of stromal CAFs in cancers. A deeper understanding of the components of the CAF secretome and their interactions with cancer cells will help in the identification of personalized biomarkers and a more precise treatment plan.

Signaling and molecular networks related to development and inflammation involved in CCA initiation and progression

Intrahepatic cholangiocarcinoma (iCCA) is a rare neoplasm of the bile ducts with a low survival rate, whose incidence is continuously increasing and is associated with a rich and varied tumor microenvironment (TME). Although the main mutations characterizing iCCA are known, there are several unresolved issues regarding the processes leading to the accumulation of mutations in the normal cholangiocyte. The inflammatory mediators and the molecular pathways involved in cholangiocarcinogenesis, which regulate the transition from normal to dysplastic cells, resulting in neoplastic cholangiocytes, are poorly understood. Moreover, once the tumor is established, it is unclear which effects of the interaction between the tumor and TME constituent cells, in particular cancer-associated fibroblasts (CAFs), are responsible for stimulating the malignant behavior of iCCA. In this review, we described the main mutations affecting the bile ducts leading to iCCA development as well as the putative inflammatory mediators and morphogenetic pathways involved in the establishment of the malignant transition of the bile ducts. We also described the main signaling pathways involved in TME-tumor cell interactions, with particular emphasis on the effect of CAFs in cancer. Finally, we wanted to analyze possible new therapeutic approaches aimed at modifying the composition of TME and the possible role of immunotherapy in improving the treatment of this cancer.

The recent advances of cancer associated fibroblasts in cancer progression and therapy.

As an abundant component of tumor microenvironment, cancer-associated fibroblasts (CAFs) are heterogeneous cell populations that play important roles in tumor development, progression and therapeutic resistance. Multiple sources of cells can be recruited and educated to become CAFs, such as fibroblasts, mesenchymal stem cells and adipocytes, which may explain the phenotypic and functional heterogeneity of CAFs. It is widely believed that CAFs regulate tumor progression by remodeling extracellular matrix, promoting angiogenesis, and releasing soluble cytokines, making them a promising cancer therapy target. In this review, we discussed about the origin, subpopulation, and functional heterogeneity of CAFs, with particular attention to recent research advances and clinical therapeutic potential of CAFs in cancer.

HNRNPA2B1 Demonstrates Diagnostic and Prognostic Values Based on Pan-Cancer Analyses.

Some studies have suggested heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) to be a promoter in cancer development. Nonetheless, no detailed pan-cancer investigation has been reported. Thus, this study explored the possible oncogenic role of HNRNPA2B1, such as its expression levels, gene alteration, protein–protein interaction network, immune infiltration, and prognostic value in different cancer types using The Cancer Genome Atlas web platform. Many types of cancer exhibit HNRNPA2B1 overexpression, which is notably associated with poor prognosis. We also found that HNRNPA2B1 with different methylation levels causes a varied prognosis in lung adenocarcinoma (LUAD). It is noteworthy that HNRNPA2B1 levels are connected with cancer-associated fibroblasts in cancers, such as adrenocortical carcinoma, LUAD, and stomach adenocarcinoma. In addition, HNRNPA2B1 participates in the spliceosome- and cell cycle-associated pathways. Finally, HNRNPA2B1 is highly valued in the diagnosis of LUAD, lung squamous cell carcinoma, breast invasive carcinoma, esophageal carcinoma, and liver hepatocellular carcinoma. This systematic study highlighted the role of HNRNPA2B1 in pan-cancer progression.

Abstract P5-06-08: Triple-negative breast cancer CAFs induce a metastatic phenotype in MCF-7 cells via the SDF-1/CXCR4 axis

Abstract Background: Metastatic disease is the foremost cause of breast cancer (BC) related mortality in women. One of the crucial challenges in reducing metastasis-related mortality is in identifying and understanding why certain BCs metastasize and recur. Although cancer cell-intrinsic factors are a key determinant in BC metastasis, host-intrinsic factors like the cells of the tumor microenvironment (TME), such as cancer-associated fibroblasts (CAFs) are what might be driving certain BCs to metastasize and recur. We recently showed that CAFs from the primary (TME) enter into circulation as cCAFs, form heterotypic clusters with tumor cells, and arrive at metastatic sites. This interaction between CAFs and tumor cells is crucial in furthering the metastatic cascade. However, the functional heterogeneity of the different CAF phenotypes and how that impacts BC metastasis is unknown. Stromal derived factor-1 (SDF-1/CXCL12) is an important chemokine that is known to be involved in promoting tumor cell invasion and metastasis. In this study, we examine the functional differences between CAF isolated from different molecular subtypes of BC. Furthermore, we elucidate the role of CAF secreted SDF-1 as being one of the mechanisms by which a subset of CAF cells permanently reprograms poorly-metastatic MCF-7 cells to augment EMT-driven genes and become metastatic in vivo. Methods: We used primary CAF cell lines derived from BCs of different molecular subtypes and examined their global gene expression profiles using RNAseq. We used NSG mice xenografted with MCF-7 BC cells and primary CAF cells to evaluate the contribution of two different CAF phenotypes in promoting BC progression and metastasis. We developed dissociated tumor cell lines from CAF co-injected MCF-7 xenografts to determine the molecular changes that CAFs impart to MCF-7 cells in vivo. Using limiting dilution tumor xenograft assays and gene expression profiles we examined the molecular pathways, BC stemness markers, and tumor-initiating capacity of the dissociated tumor cell lines. By long-term SDF-1 treatment of MCF-7 cells in vitro and using these cells in various RNASeq, in vitro, and in vivo assays we determined the role of SDF-1 in altering the phenotype of MCF-7 cells. Results: We found that CAFs from different molecular subtypes have a differential effect on tumor initiation, progression, and metastasis in xenograft assays. We also found that a subset of CAFs has the ability to make the poorly-metastatic MCF-7 cells metastatic in vivo. We also found that this subset of CAF cells has a higher expression of SDF-1 and the tumors formed with the co-injection of MCF-7 cells with this subset of CAF cells are enriched in cancer stem cell-like metastasis initiating cells. We also found that upon serial transplant, the tumors formed by CAF-reprogramed MCF-7 cells have a gene expression profile that shows enrichment for EMT-driven genes and these tumors are robustly metastatic. Furthermore, we found that the SDF-1/CXCR4 axis is one of the critical mechanisms by which this subset of CAF cells permanently reprograms the phenotype of the MCF-7 cells. Conclusions: CAFs are a highly heterogeneous population of cells in the breast TME. This study examines the contribution of the functional heterogeneity of the CAF cells in BC metastasis. We have found that a unique subset of CAF cells has the ability to reprogram a poorly metastatic BC cell to not only become metastatic but induce a permanent EMT-driven phenotypic shift in the BC cells. SDF-1 may be one of the mechanisms that drive this CAF-induced change and understanding the CAF functional landscape and its role in BC metastasis might be crucial to developing new therapeutic modalities to abrogate BC metastasis. Citation Format: Utsav Sharma, Jun Sun, Kelsie Medina-Saenz, Susan Bare, Philip Miller, Manuel Picon-Ruiz, Joyce Slingerland, Dorraya El-Ashry, Marc Lippman. Triple-negative breast cancer CAFs induce a metastatic phenotype in MCF-7 cells via the SDF-1/CXCR4 axis [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-08.

Fundamental and Clinical Applications of Materials Based on Cancer-Associated Fibroblasts in Cancers.

Cancer stromal cells play a role in promoting tumor relapse and therapeutic resistance. Therefore, the current treatment paradigms for cancers are usually insufficient to eradicate cancer cells, and anti-cancer therapeutic strategies targeting stromal cells have been developed. Cancer-associated fibroblasts (CAFs) are perpetually activated fibroblasts in the tumor stroma. CAFs are the most abundant and highly heterogeneous stromal cells, and they are critically involved in cancer occurrence and progression. These effects are due to their various roles in the remodeling of the extracellular matrix, maintenance of cancer stemness, modulation of tumor metabolism, and promotion of therapy resistance. Recently, biomaterials and nanomaterials based on CAFs have been increasingly developed to perform gene or protein expression analysis, three-dimensional (3D) co-cultivation, and targeted drug delivery in cancer treatment. In this review, we systematically summarize the current research to fully understand the relevant materials and their functional diversity in CAFs, and we highlight the potential clinical applications of CAFs-oriented biomaterials and nanomaterials in anti-cancer therapy.

Cancer-Associated Fibroblasts as Players in Cancer Development and Progression and Their Role in Targeted Radionuclide Imaging and Therapy.

Simple SummaryThe tumour microenvironment contains a unique population of cells, of complex origin and diverse functionality, known as Cancer associated fibroblasts (CAFs). In recent years there has been a growing interest in targeting CAFs to aid cancer diagnosis and therapy. Amongst the approaches utilized so far, targeting Fibroblast activation protein (FAP) has shown a lot of promise. In this review, we will focus on our current knowledge of the biology of CAFs as well as theranostic applications that will enhance cancer diagnosis and therapy in cancers carrying a distinct CAF signature.Cancer Associated Fibroblasts (CAFs) form a major component of the tumour microenvironment, they have a complex origin and execute diverse functions in tumour development and progression. As such, CAFs constitute an attractive target for novel therapeutic interventions that will aid both diagnosis and treatment of various cancers. There are, however, a few limitations in reaching successful translation of CAF targeted interventions from bench to bedside. Several approaches targeting CAFs have been investigated so far and a few CAF-targeting tracers have successfully been developed and applied. This includes tracers targeting Fibroblast Activation Protein (FAP) on CAFs. A number of FAP-targeting tracers have shown great promise in the clinic. In this review, we summarize our current knowledge of the functional heterogeneity and biology of CAFs in cancer. Moreover, we highlight the latest developments towards theranostic applications that will help tumour characterization, radioligand therapy and staging in cancers with a distinct CAF population.

Abstract 3360: Phototheranostics of cancer associated fibroblasts by targeting fibroblast activation protein-α

Abstract Introduction: Stromal cells such as cancer associated fibroblasts (CAFs) mediate many of the aggressive characteristics of cancer1 and have an ever-replenishing supply that is largely left intact by our current therapeutic strategies.2 Fibroblast activation protein-α (FAP-α) is a target antigen that been reported to be selectively expressed in tumors by a subset of immunosuppressive stromal fibroblasts. Here, we extended our previous near-infrared photoimmunotherapy (NIR-PIT) study,3 to develop a FAP-α-targeted monoclonal antibody (mAb) photosensitizer conjugate. We developed FAP-α overexpressing MDA-MB-231 and HT-1080 cells to test the feasibility of targeting FAP-α expressing populations in cells and tumors in vivo, in terms of the specificity of the conjugate to bind to, detect, and eliminate FAP-α expressing populations. Method: FAP-α monoclonal antibody (AF3715) and its IgG isotype control were conjugated with a NIR phthalocyanine dye, IR700, to form FAP-α-IR700 and IgG-IR700. FAP-α overexpression was achieved by transducing MDA-MB-231 and HT-1080 cells with lentivirus encoding the gene for human FAP (Accession No. NM_004460.3) that was subcloned into lentiviral vector pMA3211 to obtain 231-FAP and HT-1080-FAP cells. Cell viability was measured by using CCK-8 assay. Bilateral tumor models were established by inoculating 1×106 MDA-MB-231 cells or 1×106 231-FAP cells in 0.05 ml of Hank's balanced salt solution on either side in the mammary fat pads of athymic Balb/c (nu/nu) female mice. A similar number of HT-1080 or HT-1080-FAP cells were inoculated bilaterally in the flank. 50 μg of FAP-α-IR700 or IgG-IR700 was injected i.v., and fluorescence images of IR700 in mice were obtained over a 24-h period (n = 3 per group). At 24 h post injection, mice were euthanized, and tumors were isolated for imaging. For PIT, tumor-bearing mice (n = 4 per group) received two i.v. injections of 100 μg of FAP-α-IR700 at a one-week interval, and tumors were exposed to NIR irradiation at 200 J/cm2 at 24h p.i. Tumor diameters were measured over 2 weeks. IgG-IR700 and PBS-injected mice were used as controls. Results and Discussion: We confirmed FAP-α overexpression in 231-FAP and HT-1080-FAP cells. FAP-α-IR700 was activated by NIR light, causing FAP-α-specific cell death. FAP-α-IR700-mediated phototoxicity was dependent on the conjugate concentration and NIR light dose; and it was inhibited by excess AF3715. We observed the preferential accumulation of FAP-α-IR700 in FAP-α-overexpressing 231-FAP and HT-1080-FAP tumors compared to their wild-type counterparts. The mean fluorescence intensity of FAP-α-IR700 in FAP-α-overexpressing tumors was approximately two to threefold higher than the wild type tumors. FAP-α-IR700 injection together with NIR light exposure resulted in the highest tumor growth delay in 231-FAP tumors and HT-1080-FAP tumors. Our results evaluate and confirm the ability of FAP-α-IR700 to target and eliminate FAP-α-overexpressing cell populations, providing novel opportunities to selectively deplete FAP-α high CAFs in cancers.

Abstract P6-06-15: Remodelling of calcium influx pathways in breast cancer associated fibroblasts

Abstract Disruption of Ca2+ signaling during breast cancer tumorigenesis is now well-established. Despite the increasing recognition of the role of the tumour microenvironment, and cancer associated fibroblasts (CAFs), in tumorigenesis, very few studies have assessed alterations in Ca2+ influx pathways in this context. The aims of this study were to identify changes in the nature of Ca2+ influx in breast CAFs and to define the role of Ca2+ signaling in the induction of CAFs. We first developed a model for CAF induction using an immortalized human mammary fibroblast cell line, HMF3S. These cells were treated with transforming growth factor beta (TGFβ) to induce a CAF phenotype (HMF3S-CAF) as confirmed by the induction established CAF markers, including alpha smooth muscle actin. Fluorescence Imaging Plate Reader Ca2+ assays demonstrated that store operated Ca2+ entry (SOCE) was reduced as a consequence of TGFβ stimulated CAF induction in HMF3S-CAFs. As a potential mechanism, mRNA levels of the SOCE components, ORAI1, STIM1 and STIM2 were assessed. STIM2 was downregulated and ORAI1 was slightly upregulated suggesting that the decrease in SOCE in HMF3S-CAF cells were independent of mRNA expression changes of these genes. Voltage gated calcium channel (VGCC) mRNA expression was measured to determine whether other Ca2+ influx pathways were altered during CAF differentiation. Here, we found that Cav 1.2 and Cav 3.2 were significantly upregulated in HMF3S-CAF cells. Assessment of mRNA levels of SOCE components and VGCC in paired normal and CAF patient samples showed no change in SOCE components while, of the VGCCs, only Cav 1.2 and Cav 3.2 were upregulated, suggesting that increased expression of these channels may be a feature of breast cancer CAFs. The importance of Cav 1.2 and Cav 3.2 in CAF induction was assessed by pharmacological inhibition using nimodipine (Cav 1.2) or ML218 (Cav 3.2), both of which decreased TGFβ-mediated CAF activation. Our preliminary data suggest that siRNA against specific VGCCs may also reduce CAF activation. Our data indicate that Ca2+ signaling is remodelled in CAFs and that VGCC Ca2+ signal is important for breast CAF induction. Citation Format: Francisco Sadras, Teneale A Stewart, Melanie Robitaille, Amelia A Peters, Priyakshi Kalita-de Croft, Patsy Soon, Jodi M Saunus, Sunil R Lakhani, Sarah J Roberts-Thomson, Gregory R Monteith. Remodelling of calcium influx pathways in breast cancer associated fibroblasts [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-06-15.

Wnt signaling dynamics in head and neck squamous cell cancer tumor-stroma interactions.

Wnt pathway activation maintains the cancer stem cell (CSC) phenotype and promotes tumor progression, making it an attractive target for anti-cancer therapy. Wnt signaling at the tumor and tumor microenvironment (TME) front have not been investigated in depth in head and neck squamous cell carcinoma (HNSCC). In a cohort of 48 HNSCCs, increased Wnt signaling, including Wnt genes (AXIN2, LGR6, WISP1) and stem cell factors (RET, SOX5, KIT), were associated with a more advanced clinical stage. Key Wnt pathway proteins were most abundant at the cancer epithelial-stromal boundary. To investigate these observations, we generated three pairs of cancer-cancer associated fibroblast (CAF) cell lines derived from the same HNSCC patients. 3D co-culture of cancer spheres and CAFs mimicked these in vivo interactions, and using these we observed increased expression of Wnt genes (eg, WNT3A, WNT7A, WNT16) in both compartments. Of these Wnt ligands, we found Wnt3a, and less consistently Wnt16, activated Wnt signaling in both cancer cells and CAFs. Wnt activation increased CSC characteristics like sphere formation and invasiveness, which was further regulated by the presence of CAFs. Time lapse microscopy also revealed preferential Wnt activation of cancer cells. Wnt inhibitors, OMP-18R5 and OMP-54F28, significantly reduced growth of HNSCC patient-derived xenografts and suppressed Wnt activation at the tumor epithelial-stromal boundary. Taken together, our findings suggest that Wnt signaling is initiated in cancer cells which then activate CAFs, and in turn perpetuate a paracrine signaling loop. This suggests that targeting Wnt signaling in the TME is essential.

Development of an in vitro cell-sheet cancer model for chemotherapeutic screening

Epithelial cancer grows in vivo in a microenvironment that comprises tumour, stroma, and immune cells. A three-dimensional (3D) culture model might be able to mimic the tumour microenvironment in vivo; therefore, we developed a new 3D epithelial cancer model using in vitro cell-sheet engineering and compared the results of treatment with several chemotherapeutic drugs among the 3D cell-sheet model, spheroid culture, and 2D cell culture.Methods: The cell sheet comprised keratinocytes and a plasma fibrin matrix containing fibroblasts. Cancer spheroids with or without cancer-associated fibroblasts (CAFs) were interposed between the keratinocytes and fibrin layer. Cell growth, viability, and hypoxia were measured using the cell counting kit-8, LIVE/DEAD assay, and propidium iodide and LOX-1 staining. The morphology, invasion, and mRNA and protein expression were compared among the different cell culture models.Results: Enhanced resistance to sorafenib and cisplatin by cancer spheroids and CAFs was more easily observed in the 3D than in the 2D model. Invasion by cancer-CAF spheroids into the fibrin matrix was more clearly observed in the 3D cell sheet. The expansion of viable cancer cells increased in the 3D cell sheet, particularly in those with CAFs, which were significantly inhibited by treatment with 10 μM sorafenib or 20 μM cisplatin (P < 0.05). TGF-β1, N-cadherin, and vimentin mRNA and protein levels were higher in the 3D cell-sheet model.Conclusions: The 3D cell sheet-based cancer model could be applied to in vitro observation of epithelial cancer growth and invasion and to anticancer drug testing.

Highlights of This Issue

The evidence that aspirin prevents cancer is compelling; however, the underlying mechanism leading to its anti-cancer effect is enigmatic. In this research article, cyclin A2 and CDK2 are demonstrated to be novel targets of aspirin and its primary metabolite, salicylic acid. Both therapeutic agents caused down-regulation of cyclin A2 and CDK2, at the protein and mRNAs levels, in a diverse panel of cancer cells and resulted in a reduction in CDK2 activity. Importantly, it is also demonstrated that CDK2 is a novel salicylic acid binding protein. These results show that the anti-cancer effects of aspirin occur through down-regulation of cell cycle regulatory proteins.Multiple myeloma resides in a hypoxic environment and shows signs of an induced hypoxic response that contributes to tumorigenesis and tumor drug resistance. Targeting this adaptive hypoxic response would have important ramifications for treating this disease. To test this, Mysore and colleagues used a Pyrrole-Imidazole polyamide that can displace a major regulator of hypoxic response, HIF1, from its DNA-binding site. Evidence shows that the polyamide suppressed growth of multiple myeloma xenografts in mice and inhibited the hypoxic response in vitro and in vivo; thus, revealing the therapeutic utility of these molecules in the treatment of multiple myeloma.Pancreatic cancer is a deadly and highly metastatic disease frequently harboring activating mutations in the KRAS oncogene. MicroRNAs (miRs) are important regulators of gene expression with an established role in cancer pathogenesis. Here, miR-31 is identified as a transcriptional target of the RAS-MAPK signaling pathway. The miR-31 host gene promoter is activated by the transcription factor ELK1. Enforced expression of miR-31 enhanced invasion-migration of pancreatic cancer cells via down regulation of the miR-31 target RASA1 and activation of RHOA. These results implicate miR-31 as a novel effector in the RAS-transformation program and demonstrate a mechanism for the metastatic behavior of pancreatic cancer.Cancer-associated fibroblasts (CAFs) are a vital cellular component of the tumor microenvironment (TME) and are a recognized source of intratumoral collagen. This study is important because it shows that CAFs not only produce collagen but also regulate the types of cross-links that form between collagen strands and contribute to the biomechanical properties of tumor stroma. Mechanistically, this activity of CAFs was mediated by lysyl hydroxylase 2 (PLOD2/LH2), which strengthens the growing body of evidence that targeting LH2 has the potential to suppress the growth and metastasis of tumors in patients.