bims-tuchim Biomed News
on Tumor-on-chip models
Issue of 2021‒03‒07
ten papers selected by
Philipp Albrecht
Friedrich Schiller University

  1. Proc Natl Acad Sci U S A. 2021 Mar 09. pii: e2024780118. [Epub ahead of print]118(10):
      Contact guidance is a major physical cue that modulates cancer cell morphology and motility, and is directly linked to the prognosis of cancer patients. Under physiological conditions, particularly in the three-dimensional (3D) extracellular matrix (ECM), the disordered assembly of fibers presents a complex directional bias to the cells. It is unclear how cancer cells respond to these noncoherent contact guidance cues. Here we combine quantitative experiments, theoretical analysis, and computational modeling to study the morphological and migrational responses of breast cancer cells to 3D collagen ECM with varying degrees of fiber alignment. We quantify the strength of contact guidance using directional coherence of ECM fibers, and find that stronger contact guidance causes cells to polarize more strongly along the principal direction of the fibers. Interestingly, sensitivity to contact guidance is positively correlated with cell aspect ratio, with elongated cells responding more strongly to ECM alignment than rounded cells. Both experiments and simulations show that cell-ECM adhesions and actomyosin contractility modulate cell responses to contact guidance by inducing a population shift between rounded and elongated cells. We also find that cells rapidly change their morphology when navigating the ECM, and that ECM fiber coherence modulates cell transition rates between different morphological phenotypes. Taken together, we find that subcellular processes that integrate conflicting mechanical cues determine cell morphology, which predicts the polarization and migration dynamics of cancer cells in 3D ECM.
    Keywords:  cell motility; contact guidance; extracellular matrix; phenotypic transition
  2. Front Bioeng Biotechnol. 2020 ;8 611802
      Metastasis is the primary cause of a large number of cancer-associated deaths. By portraying the precise environment of the metastasis process in vitro, the microfluidic system provides useful insights on the mechanisms underlying cancer cell migration, invasion, colonization, and the procurement of supplemental nutrients. However, current in vitro metastasis models are biased in studying blood vessel-based metastasis pathways and thus the understanding of lymphatic metastasis is limited which is also closely related to the inflammatory system. To understand the effects of inflammatory cytokines in lymphatic metastasis, we developed a three-channel microfluidic system by mimicking the lymph vessel-tissue-blood vessel (LTB) structure. Based on the LTB chip, we successfully confirmed the inflammatory cytokine, interleukin 6 (IL-6), -mediated intercellular communication in the tumor microenvironment during lymphatic metastasis. The IL-6 exposure to different subtypes of breast cancer cells was induced epithelial-mesenchymal transition (EMT) and improved tissue invasion property (8-fold). And the growth of human vein endothelial cells toward the lymph vessel channel was observed by VEGF secretion from human lymphatic endothelial cells with IL-6 treatment. The proposed LTB chip can be applied to analyze the intercellular communication during the lymphatic metastasis process and be a unique tool to understand the intercellular communication in the cancer microenvironment under various extracellular stimuli such as inflammatory cytokines, stromal reactions, hypoxia, and nutrient deficiency.
    Keywords:  angiogenesis; cancer metastasis; circulating tumor cells; epithelial-mesenchymal transition; lymph vessel; microfluidics
  3. Mol Ther. 2021 Feb 26. pii: S1525-0016(21)00127-1. [Epub ahead of print]
      Chimeric antigen receptor (CAR) T cell therapy has led to impressive clinical responses in patients with hematological malignancies; however, its effectiveness in patients with solid tumors has been limited. While CAR T cells for the treatment of advanced prostate and pancreas cancer, including those targeting prostate stem cell antigen (PSCA), are being clinically evaluated and are anticipated to show bioactivity, their safety and the impact of the immunosuppressive tumor microenvironment (TME) have not been faithfully explored preclinically. Using a novel human PSCA knock-in (hPSCA-KI) immunocompetent mouse model, we evaluate safety and therapeutic efficacy of PSCA-CAR T cells. We demonstrated that cyclophosphamide (Cy) pre-conditioning significantly modified the immunosuppressive TME and was required to uncover the efficacy of PSCA-CAR T cells in metastatic prostate and pancreas cancer models, with no observed toxicities in normal tissues with endogenous expression of PSCA. This combination dampened the immunosuppressive TME, generated pro-inflammatory myeloid and T cell signatures in tumors, and enhanced the recruitment of antigen-presenting cells as well as endogenous and adoptively-transferred T cells, resulting in long-term anti-tumor immunity.
    Keywords:  adoptive cellular immunotherapy; chimeric antigen receptor; cyclophosphamide; immunosuppression; pancreatic cancer; pre-conditioning; prostate cancer; prostate stem cell antigen; tumor microenvironment
  4. Biomed Pharmacother. 2021 Mar 02. pii: S0753-3322(21)00221-3. [Epub ahead of print]138 111436
      Triple-negative breast cancer (TNBC) is an invasive tumor with a high incidence of distant metastasis and poor prognosis. In TNBC cells, high PD-L1 expression can induce an immunosuppressive tumor microenvironment, repressing the anti-tumoral immune responses. Although FDA-approved agents targeting the PD-1/PD-L1 axis are potent to eliminate tumoral cells, their immune-related adverse events have become worrisome. As the regulator of gene expression, siRNAs can directly target PD-L1 in breast cancer cells. The gene modification of tumoral PD-L1 can reduce our reliance on the current method of targeting the PD-L1/PD-1 axis. We initiated the study with bioinformatics analysis; the results indicated that TNBC and the MDA-MB-231 cells significantly overexpressed PD-L1 compared to other breast cancer subtypes and cell lines. Our results demonstrated that PD-L1 silencing substantially reduced PD-L1 expression at mRNA and protein levels in MDA-MB-231 cells. Moreover, our results demonstrated that PD-L1 knockdown reduced cancer cell proliferation and induced apoptosis via intrinsic and extrinsic apoptosis pathways. We observed that PD-L1 silencing effectively inhibited the migration of TNBC cells. Further investigation also displayed that silencing of PD-L1 in breast cancer cells induced T-cell cytotoxic function by upregulating the gene expression of pro-inflammatory cytokines, i.e., IL-2, IFN-γ, and TNF-α, and downregulating the gene expression of anti-inflammatory cytokines, i.e., IL-10, and TGF-β, in a co-culture system.
    Keywords:  PD-L1; SiRNA; Silencing; Triple-negative breast cancer
  5. Cancer Cell. 2021 Feb 25. pii: S1535-6108(21)00109-4. [Epub ahead of print]
      Stromal desmoplastic reaction in pancreatic ductal adenocarcinoma (PDAC) involves significant accumulation of type I collagen (Col1). However, the precise molecular and mechanistic contribution of Col1 in PDAC progression remains unknown. Activated pancreatic stellate cells/αSMA+ myofibroblasts are major contributors of Col1 in the PDAC stroma. We use a dual-recombinase genetic mouse model of spontaneous PDAC to delete Col1 specifically in myofibroblasts. This results in significant reduction of total stromal Col1 content and accelerates the emergence of PanINs and PDAC, decreasing overall survival. Col1 deletion leads to Cxcl5 upregulation in cancer cells via SOX9. Increase in Cxcl5 is associated with recruitment of myeloid-derived suppressor cells and suppression of CD8+ T cells, which can be attenuated with combined targeting of CXCR2 and CCR2 to restrain accelerated PDAC progression in the setting of stromal Col1 deletion. Our results unravel the fundamental role of myofibroblast-derived Co1l in regulating tumor immunity and restraining PDAC progression.
    Keywords:  B cells; T cells; extracellular matrix; fibroblasts; genetically engineered mouse models; myeloid-derived suppressor cells (MDSCs); pancreatic ductal adenocarcinoma (PDAC); tumor immunology; tumor microenvironment; type I collagen
  6. Int J Mol Sci. 2021 Feb 14. pii: 1908. [Epub ahead of print]22(4):
      With increasing global health threats has come an urgent need to rapidly develop and deploy safe and effective therapies. A common practice to fast track clinical adoption of compounds for new indications is to repurpose already approved therapeutics; however, many compounds considered safe to a specific application or population may elicit undesirable side effects when the dosage, usage directives, and/or clinical context are changed. For example, progenitor and developing cells may have different susceptibilities than mature dormant cells, which may yet be different than mature active cells. Thus, in vitro test systems should reflect the cellular context of the native cell: developing, nascent, or functionally active. To that end, we have developed high-throughput, two- and three-dimensional human induced pluripotent stem cell (hiPSC)-derived neural screening platforms that reflect different neurodevelopmental stages. As a proof of concept, we implemented this in vitro human system to swiftly identify the potential neurotoxicity profiles of 29 therapeutic compounds that could be repurposed as anti-virals. Interestingly, many compounds displayed high toxicity on early-stage neural tissues but not on later stages. Compounds with the safest overall viability profiles were further evaluated for functional assessment in a high-throughput calcium flux assay. Of the 29 drugs tested, only four did not modulate or have other potentially toxic effects on the developing or mature neurospheroids across all the tested dosages. These results highlight the importance of employing human neural cultures at different stages of development to fully understand the neurotoxicity profile of potential therapeutics across normal ontogeny.
    Keywords:  drug discovery; human induced pluripotent stem cells (hiPSCs); neurodevelopmental toxicity; organoids; repurposed drugs
  7. Nat Biotechnol. 2021 Mar 01.
      Peptide-major histocompatibility complex (pMHC) multimers enable the detection of antigen-specific T cells in studies ranging from vaccine efficacy to cancer immunotherapy. However, this technology is unreliable when applied to pMHC class II for the detection of CD4+ T cells. Here, using a combination of molecular biological and immunological techniques, we cloned sequences encoding human leukocyte antigen (HLA)-DP, HLA-DQ and HLA-DR molecules with enhanced CD4 binding affinity (with a Kd of 8.9 ± 1.1 µM between CD4 and affinity-matured HLA-DP4) and produced affinity-matured class II dimers that stain antigen-specific T cells better than conventional multimers in both in vitro and ex vivo analyses. Using a comprehensive library of dimers for HLA-DP4, which is the most frequent HLA allele in many ancestry groups, we mapped 103 HLA-DP4-restricted epitopes derived from diverse tumor-associated antigens and cloned the cognate T-cell antigen receptor (TCR) genes from in vitro-stimulated CD4+ T cells. The availability of affinity-matured class II dimers across HLA-DP, HLA-DQ and HLA-DR alleles will aid in the investigation of human CD4+ T-cell responses.
  8. Int J Mol Sci. 2021 Feb 12. pii: 1827. [Epub ahead of print]22(4):
      T cell immunotherapy is now a mainstay therapy for several blood-borne cancers as well as metastatic melanoma. Unfortunately, many epithelial tumors respond poorly to immunotherapy, and the reasons for this are not well understood. Cancer-associated fibroblasts (CAFs) are the most frequent non-neoplastic cell type in most solid tumors, and they are emerging as a key player in immunotherapy resistance. A range of immortalized CAF lines will be essential tools that will allow us to understand immune responses against cancer and develop novel strategies for cancer immunotherapy. To study the effect of CAFs on T cell proliferation, we created and characterized a number of novel immortalized human CAFs lines (Im-CAFs) from human breast, colon, and pancreatic carcinomas. Im-CAFs shared similar phenotypes, matrix remodeling and contraction capabilities, and growth and migration rates compared to the primary CAFs. Using primary isolates from breast carcinoma, colorectal carcinoma, and pancreatic ductal adenocarcinoma, we report that CAFs across major tumor types are able to potently suppress T cell proliferation in vitro. Im-CAFs retained this property. Im-CAFs are a key tool that will provide important insights into the mechanisms of CAF-mediated T cell suppression through techniques such as CRISPR-Cas9 modification, molecular screens, and pipeline drug testing.
    Keywords:  Stromal cells; T cells; cancer-associated fibroblasts; tumor immunology; tumor microenvironment
  9. Nat Protoc. 2021 Mar 05.
      Advanced in vitro kidney models are of great importance to the study of renal physiology and disease. Kidney tubuloids can be established from primary cells derived from adult kidney tissue or urine. Tubuloids are three-dimensional multicellular structures that recapitulate tubular function and have been used to study infectious, malignant, metabolic, and genetic diseases. For tubuloids to more closely represent the in vivo kidney, they can be integrated into an organ-on-a-chip system that has a more physiological tubular architecture and allows flow and interaction with vasculature or epithelial and mesenchymal cells from other organs. Here, we describe a detailed protocol for establishing tubuloid cultures from tissue and urine (1-3 weeks), as well as for generating and characterizing tubuloid cell-derived three-dimensional tubular structures in a perfused microfluidic multi-chip platform (7 d). The combination of the two systems yields a powerful in vitro tool that better recapitulates the complexity of the kidney tubule with donor-specific properties.
  10. Bio Protoc. 2020 Jul 05. 10(13): e3675
      Pluripotent stem cells (PSCs) have the potential to provide homogeneous cell populations of T cells that can be grown at a clinical scale and genetically engineered to meet specific clinical needs. OP9-DLL4, a stromal line ectopically expressing the Notch ligand Delta-like 4 (DLL4) is used to support differentiation of PSCs to T-lymphocytes. This article outlines several protocols related to generation of T cells from human and non-human primate (NHP) PSCs, including initial hematopoietic differentiation of PSC on OP9 feeders or defined conditions, followed by coculture of the OP9-DLL4 cells with the PSC-derived hematopoietic progenitors (HPs), leading to efficient differentiation to T lymphocytes. In addition, we describe a protocol for robust T cell generation from hPSCs conditionally expressing ETS1. The presented protocols provide a platform for T cell production for disease modeling and evaluating their use for immunotherapy in large animal models.
    Keywords:  Hematopoietic differentiation; Hematopoietic progenitor; Hemogenic endothelium; Human embryonic stem cells; Human pluripotent stem cells; Non-human primate pluripotent stem cells; T cells