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Introduction: Breast cancer is one of the most frequently diagnosed cancer in women with a high mortality rate. More than 20 different subtypes of breast cancer are identified. Advancement in patient-derived organoid technology makes it possible to preserve cellular, structural, and tissue microenvironment which mimics the tissue in vivo. Method: We developed patient derived breast organoids both from normal and cancer biopsies recapitulate the structure of breast tissue. Breast biopsies were enzymatically digested to yield a single cell suspension. About 1 × 105 cells were encapsulated in a specialized hydrogel mixture. Immune enhanced breast tumor organoids (iTOs) included 2 × 105 patient matched PMBCs. Results: Histological analyses of the breast organoids shows the characteristics of the breast tissue with well-defined acini in H&E. In tumor organoids acini were somewhat perturbed compared to normal breast organoids. Immuno-fluorescence staining shows the expression of breast biomarkers including EGF receptor 2 (HER2), Progesterone receptor (PR), Estrogen receptor (ER). Zona occludin 1 and 2 and keratin 19 expression in luminal cells and expression of Keratin 14 and P63 in basal cells suggested correct polarization in the organoids. Immunofluorescence staining of (iTOs), with T cell markers including CD3, CD4, and CD8 indicated that immune cells remained viable in the iTOs. Drug responses to Doxorubicin, Paclitaxel and a combination of Doxorubicin-Paclitaxel showed significant inhibition of cell growth in normal and tumor organoids (p< 0.04, n=11) except for Paclitaxel failed to inhibit tumor cell growth. However, immunotherapy with nivolumab (anti PD-1) showed no significant effect on cell growth. Conclusions: Pateint Derived Breast cancer organoids recapitulate the histological features of breast tissue in culture and response to chemotherapies. In the future, patient-derived tumor organoids can provide a platform for personalized medicine. Citation Format: Nadeem Wajih, Richard Erali, Steven Forsythe, Shay Soker, Konstantinos Votanopoulos. Pateint Derived Breast Cancer Organoids as a Model for Testing Personalized Therapies [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-09-02.

supplemental materials and methods and supplemental figures

Metastasis is the main cause of cancer-related mortality. Despite intense efforts to understand the mechanisms underlying the metastatic process, treatment of metastatic cancer is still challenging. Here we describe a chemotherapy-induced, host-mediated mechanism that promotes remodeling of the extracellular matrix (ECM), ultimately facilitating cancer cell seeding and metastasis. Paclitaxel (PTX) chemotherapy enhanced rapid ECM remodeling and mechanostructural changes in the lungs of tumor-free mice, and the protein expression and activity of the ECM remodeling enzyme lysyl oxidase (LOX) increased in response to PTX. A chimeric mouse model harboring genetic LOX depletion revealed chemotherapy-induced ECM remodeling was mediated by CD8+ T cells expressing LOX. Consistently, adoptive transfer of CD8+ T cells, but not CD4+ T cells or B cells, from PTX-treated mice to naïve immunodeprived mice induced pulmonary ECM remodeling. Lastly, in a clinically relevant metastatic breast carcinoma model, LOX inhibition counteracted the metastasis-promoting, ECM-related effects of PTX. This study highlights the role of immune cells in regulating ECM and metastasis following chemotherapy, suggesting that inhibiting chemotherapy-induced ECM remodeling represents a potential therapeutic strategy for metastatic cancer.Significance:Chemotherapy induces prometastatic pulmonary ECM remodeling by upregulating LOX in T cells, which can be targeted with LOX inhibitors to suppress metastasis.See related commentary by Kolonin and Woodward, p. 197

Purpose:Immunotherapy efficacy data on appendiceal cancer from clinical trials does not exist, due to appendiceal cancer incidence of 0.97 per 100,000. The goal of this study was to preclinically explore the application of immunotherapy in treating appendiceal cancer in a personalized organoid model.Experimental Design:Patient tumor organoids (PTO) were fabricated using unsorted tumor cells with and without enrichment with patient-matched immune components derived from peripheral blood leukocytes, spleen, or lymph nodes [immune-enhanced PTOs (iPTO)]. Organoids were cultured for 7 days, followed by treatment with immunotherapy (pembrolizumab, ipilimumab, nivolumab), and assessed for treatment efficacy.Results:Between September 2019 and May 2021, 26 patients were enrolled in the study. Successful testing was conducted in 19 of 26 (73.1%) patients, with 13 of 19 (68.4%) and 6 of 19 (31.6%) patients having low-grade appendiceal (LGA) and high-grade appendiceal (HGA) primaries, respectively. Immunotherapy response, with increased expression of granzyme B and cleaved caspase 3 and decreased expression of CK20 and ATP activity, was exhibited in 4 of 19 (21.1%) pembrolizumab-treated and 2 of 19 (10.5%) nivolumab-treated iPTOs. Post-immunotherapy cellular viability, in responding HGA organoids to pembrolizumab, decreased to less than 15% (P < 0.05). LGA iPTO treatment responses were observed in pembrolizumab and nivolumab, with an 8%–47.4% (P < 0.05) viability compared with controls. Ipilimumab showed no efficacy in the examined cohort.Conclusions:Immunotherapy shows measurable efficacy in appendiceal cancer organoids. Information derived from immunocompetent organoids may be applied in selecting patients for clinical trial enrollment in rare diseases where preclinical models of disease are lacking.

Flow cytometry analysis of CD8+ T-cells after co-culture with tumor cells in organoids for 1 week.

Three-dimensional (3D) cell culture systems such as organoids are the cumulative construction of advances in the fields of biomaterials, biofabrication, tissue engineering, and cellular and tissue physiology. These organoids represent a more realistic model than their two-dimensional (2D) cell culture counterparts. They are able to maintain the natural morphology of cellular systems through increased intercellular contact, extracellular matrix (ECM) formation, and addition and support for innate signaling mechanisms not replicated in 2D models. There are a number of different fabrication methods and specifications currently used to create 3D culture systems. The variation in methods results in diverse systems that specialize in modeling of equally diverse in vivo systems, including specific cancer types and immune environments. The ability to readily create physiologically relevant tissue equivalents in vitro with a similar tumor microenvironment (TME) to a patient's tumor tissue, including both cancer and stromal components, greatly increases the scope of precision medicine and can allow for more accurate analysis of therapeutics and cell behavior.

Metastasis is the main cause of cancer-related mortality. Despite intense efforts to understand the mechanisms underlying the metastatic process, treatment of metastatic cancer is still challenging. Here we describe a chemotherapy-induced, host-mediated mechanism that promotes remodeling of the extracellular matrix (ECM), ultimately facilitating cancer cell seeding and metastasis. Paclitaxel (PTX) chemotherapy enhanced rapid ECM remodeling and mechanostructural changes in the lungs of tumor-free mice, and the protein expression and activity of the ECM remodeling enzyme lysyl oxidase (LOX) increased in response to PTX. A chimeric mouse model harboring genetic LOX depletion revealed chemotherapy-induced ECM remodeling was mediated by CD8+ T cells expressing LOX. Consistently, adoptive transfer of CD8+ T cells, but not CD4+ T cells or B cells, from PTX-treated mice to naïve immunodeprived mice induced pulmonary ECM remodeling. Lastly, in a clinically relevant metastatic breast carcinoma model, LOX inhibition counteracted the metastasis-promoting, ECM-related effects of PTX. This study highlights the role of immune cells in regulating ECM and metastasis following chemotherapy, suggesting that inhibiting chemotherapy-induced ECM remodeling represents a potential therapeutic strategy for metastatic cancer. Significance: Chemotherapy induces prometastatic pulmonary ECM remodeling by upregulating LOX in T cells, which can be targeted with LOX inhibitors to suppress metastasis. See related commentary by Kolonin and Woodward, p. 197

The fields of regenerative medicine and tissue engineering offer new therapeutic options to restore, maintain or improve tissue function following disease or injury. To maximize the biological function of a tissue-engineered clinical product, specific conditions must be maintained within a bioreactor to allow the maturation of the product in preparation for implantation. Specifically, the bioreactor should be designed to mimic the mechanical, electrochemical and biochemical environment that the product will be exposed to in vivo. Real-time monitoring of the functional capacity of tissue-engineered products during manufacturing is a critical component of the quality management process. The present review provides a brief overview of bioreactor engineering considerations. In addition, strategies for bioreactor automation, in-line product monitoring and quality assurance are discussed.

Introduction: Tumor infiltrating lymphocytes (TILs), hold promise in advancing adoptive cell therapy for patients with otherwise limited treatment options. However, many tumors do not attract TILs, or the TILs themselves are exhausted which limits treatment availability and efficacy. Consequently, there is a need for alternative sources of tumor reactive T cells. Here, we address this need through the ex vivo 3D cell culture biofabrication of immune competent patient tumor organoids (iPTOs). These constructs, composed of patient-matched cancer cells, antigen presenting cells (APCs), and stromal cells encapsulated in extracellular matrix (ECM)-like hydrogel, closely mimic the in vivo tumor microenvironment. We demonstrate that constant circulation of peripheral blood mononuclear cells (PBMCs) through a microfluidic device housing iPTOs can emulate lymph node activation of immune cells to the tumor, yielding organoid interacting lymphocytes (OILs) that possess increased markers of activation, cytotoxicity, proliferation, homing markers, and inflammatory cytokine production relative to uncirculated PBMCs. Methods: Tumor tissue, APCs (from lymph nodes or spleen), and peripheral blood were collected from 8 patients with mesothelioma (3), melanoma (2), and appendiceal cancer (3). Chips were built from a glass slide and laser cut Polymethyl methacrylate. IPTOs were made such that a concentration of 3 APCs for every 1 tumor cell were added to a collagen and hyaluronic acid mixture that was then photocrosslinked in the chip chamber. PBMCs were circulated through chips housing the photocrosslinked iPTOs for 7 days and then expanded. Following expansion, a subpopulation of these cells were cocultured with tumor only organoids for a 7 day period. Single and secondary tumor exposure populations of effector, memory, and reactive T cells were analyzed via flow cytometry, immunohistochemistry, and cell culture medium proteomic analysis, and compared with both uncirculated PBMCs and patient TILs. Results: Data collected from patients with appendiceal, melanoma, and mesothelioma tumors shows generation of OILS with increase in cytotoxic T lymphocyte, effector memory, and central memory phenotypes when compared to uncirculated PBMCs, greater than or comparable to TILs and with similar effector cytokine and enzyme expression. OILs were also found to express less immunosuppressive signals (i.e., Tim3, PD1, and IL 10). Critically, chip-based T cell activation resulted in 10x more viable OILs than were typically observed with TILs following parallel growth period of 14 days. Conclusions: This data suggests that microfluidic incubation of lymphocytes with lymph node and tumor cells can generate large amounts of viable tumor-specific T cells on-demand with characteristics similar to TILs but with increased viability, cell expansion propensity, and cytotoxic responsiveness. Citation Format: Damian C. Hutchins, Cecilia R. Schaaf, Nicholas P. Edenhoffer, Mitra Kooshki, Robyn Greissenger, Steven D. Forsythe, Adam R. Hall, Lance D. Miller, Shay Soker, Pierre L. Triozzi, Konstantinos Votanopoulos. Microfluidic incubation of patient derived tumor and immune cells boosts lymphocyte cytotoxic phenotype. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4051.

Introduction: Patient tumor organoids (PTOs) are a new and developing preclinical model in cancer research. However, there is variation in protocols between centers and lack of consistency in important variables, impacting PTO culture success. In this study, we analyzed factors in clinical data and tumor tissue processing to determine optimal conditions for cell isolation and PTO success. Methods: Under IRB approval, tumor tissues from primary and metastatic sites were procured in clinically indicated surgeries then dissociated using a standardized protocol. PTOs were fabricated using collagen-hyaluronic acid ECM and underwent drug screening at 10 days post fabrication, where viability was assessed. Clinical and tissue processing inputs such as type of primary, peritoneal carcinomatosis index (PCI), prior surgery or neoadjuvant chemotherapy, presence of mucin, tissue digestion time, specimen mass, digestion time, and the diameter of isolated cells were analyzed. These factors were assessed by calculating the Pearson Correlation Coefficient (r) and best fit modeling to determine the effect on outputs including tissue viability as measured by % of viable cells using an automated cellular counter, viable cells per mg tissue, and PTO log10 viability by Promega 3D Cell Titer Glo assay. Results: From December 2018-July 2022, 515 tumors from 286 patients with 25 primary types were processed for cellular isolation and PTO fabrication. 407 (79%) provided PTOs for therapeutic testing, with 354 (69%) providing sufficiently viable PTOs for therapeutic interrogation. The most common primary tumor type accrued was appendiceal cancer (n=223, 43%). Tissues processed at 24 hours did not differ in viability outputs when compared to tissues processed immediately (p>0.05). Tissue % viability was better maintained in larger specimens (r=.18, p Conclusions: Higher rates of successful PTO fabrication are obtained in patients with untreated tumors, more aggressive biologic behavior, and increased tumor burden. Creating a unified protocol for tissue processing, agnostic to tumor type, will facilitate incorporation of tissue from the operating room to research protocols with wider translational implications. Citation Format: Steven Donald Forsythe, Richard A. Erali, Preston Laney, William Meeker, Salomat Abdulkhuseynova, Nadeem Wajih, Nicholas Edenhoffer, Cecilia R. Schaaf, Hemamyammal Sivakumar, Aleksander Skardal, Roy E. Strowd, Stephen B. Tatter, Clancy J. Clark, Reese W. Randle, Perry Shen, Edward A. Levine, Ralph B. D'Agostino, Shay Soker, Konstantinos I. Votanopoulos. A 515 tumor specimens collection from surgery to benchtop: the WFORCE organoid experience in personalized research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 150.

Introduction: Breast cancer is one of the most frequently diagnosed cancers among women, with a high mortality rate. More than 20 different subtypes of breast cancer are identified. Advancement in patient-derived organoid technology makes it possible to preserve cellular, structural, and tissue microenvironment, which mimics the tissue in vivo. The present study aims to develop a cryopreserved (living) tumor biobank from patient-derived tumor breast tissues. Eventually, biobank organoids will be used to test treatment options outside of patients, in a dish, and provide information on what treatments the patient responds to. Method: Patient-derived organoids (PDOs) were bio-fabricated using an unsorted tumor and normal cell suspension in a collagen-based hydrogel. Immune-enhanced tumor organoids (iTOs) were also bio-fabricated using patient-matched PMBCs and tumor cells. Breast cancer living biobanks were developed by freezing organoids in cryopreservation solutions and reestablishing them in growth media compatible with breast organoids. Histological staining for hormone receptors and Her2 receptor markers was performed. Organoids were cultured for seven days, followed by treatment with chemotherapy (Paclitaxel, Doxorubicin, and Sulphonamide) and immunotherapy (Nivolumab, Pembrolizumab, and Atezolizumab), and assessed for cell viability. Results: H&E staining showed the characteristics of the breast tissue with well-defined acini. In tumor organoids, acini were somewhat perturbed compared to normal breast organoids. Immunofluorescence staining showed the expression of breast biomarkers, including EGF receptor 2 (HER2), Progesterone receptor (PR), and Estrogen receptor (ER). Zona occludin 2 and keratin 19 expression in luminal cells and expression of Keratin 14 and P63 in basal cells, suggesting correct polarization in the organoids. Immunofluorescence staining of iTOs, with T cell markers including CD3, CD4, and CD8, indicated that immune cells remained viable in the iTOs. Drug responses to Doxorubicin, Paclitaxel, Sulphonamide and a combination of Doxorubicin-Paclitaxel showed significant inhibition of cell growth in normal and tumor organoids (p< 0.04). Treatment of breast iTOs showed moderate responses to nivolumab (n=11) and pembrolizumab (n=5), with a 63%-100% efficacy (P < 0.05) respectively. No responses were observed for Atezolizumab (n=5). Drug responses of biobank organoids showed comparable drug sensitivities to their fresh, non-frozen counterparts, suggesting that biobank organoids retain physiological relevant information and can be used for testing new drug therapies. Conclusion: Breast cancer organoids recapitulate the histological features of breast tissue in culture and respond to chemotherapy and immunotherapy. In the future, patient-derived breast tumor organoids can be cryopreserved and serve as living biobanks to provide a platform for personalized medicine. Citation Format: Nadeem Wajih, Richard Erali, Edward A. Levine, Konstantinos Votanopoulos, Shay Soker. Development of a living biobank of breast cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 164.

Colorectal cancer (CRC) treatment is currently hindered by micrometastatic relapse that cannot be removed completely during surgery and is often chemotherapy resistant. Targeted theranostic nanoparticles (NPs) that can produce heat for ablation and enable tumor visualization via their fluorescence offer advantages for detection and treatment of disseminated small nodules. A major hurdle in clinical translation of nanoparticles is their interaction with the 3D tumor microenvironment. To address this problem tumor organoid technology was used to evaluate the ablative potential of CD44-targeted polymer nanoparticles using hyaluronic acid (HA) as the targeting agent and coating it onto hybrid donor acceptor polymer particles (HDAPPs) to form HA-HDAPPs. Additionally, nanoparticles composed from only the photothermal polymer, poly[4,4-bis(2-ethylhexyl)-cyclopenta[2,1-b;3,4-b’]dithiophene-2,6-diyl-alt-2,1,3-benzoselenadiazole-4,7-diyl] (PCPDTBSe), were also coated with HA, to form HA-BSe NPs, and evaluated in 3D. Monitoring of nanoparticle transport in 3D organoids revealed uniform diffusion of non-targeted HDAPPs in comparison to attenuated diffusion of HA-HDAPPs due to nanoparticle-matrix interactions. Computational diffusion profiles suggested that HA-HDAPPs transport may not be accounted for by diffusion alone, which is indicative of nanoparticle/cell matrix interactions. Photothermal activation revealed that only HA-BSe NPs were able to significantly reduce tumor cell viability in the organoids. Despite limited transport of the CD44-targeted theranostic nanoparticles, their targeted retention provides increased heat for enhanced photothermal ablation in 3D, which is beneficial for assessing nanoparticle therapies prior to in vivo testing.

BACKGROUND. Chemotherapy dosing duration and perfusion temperature vary significantly in HIPEC protocols. This study investigates patient-derived tumor organoids as a platform to identify the most efficacious perfusion protocol in a personalized approach. PATIENTS AND METHODS. Peritoneal tumor tissue from 15 appendiceal and 8 colon cancer patients who underwent CRS/HIPEC were used for personalized organoid development. Organoids were perfused in parallel at 37 and 42 °C with low- and high-dose oxaliplatin (200 mg/m(2) over 2 h vs. 460 mg/m(2) over 30 min) and MMC (40 mg/3L over 2 h). Viability assays were performed and pooled for statistical analysis. RESULTS. An adequate organoid number was generated for 75% (6/8) of colon and 73% (11/15) of appendiceal patients. All 42 °C treatments displayed lower viability than 37 °C treatments. On pooled analysis, MMC and 200 mg/m(2) oxaliplatin displayed no treatment difference for either appendiceal or colon organoids (19% vs. 25%, p = 0.22 and 27% vs. 31%, p = 0.55, respectively), whereas heated MMC was superior to 460 mg/m(2) oxaliplatin in both primaries (19% vs. 54%, p < 0.001 and 27% vs. 53%, p = 0.002, respectively). In both appendiceal and colon tumor organoids, heated 200 mg/m(2) oxaliplatin displayed increased cytotoxicity as compared with 460 mg/m(2) oxaliplatin (25% vs. 54%, p < 0.001 and 31% vs. 53%, p = 0.008, respectively). CONCLUSIONS. Organoids treated with MMC or 200 mg/m(2) heated oxaliplatin for 2 h displayed increased susceptibility in comparison with 30-min 460 mg/m(2) oxaliplatin. Optimal perfusion protocol varies among patients, and organoid technology may offer a platform for tailoring HIPEC conditions to the individual patient level.

Liver fibrosis occurs in most cases of chronic liver disease, which are somewhat common, but also a potentially deadly group of diseases. In vitro modeling of liver fibrosis relies primarily on the isolation of in vivo activated hepatic stellate cells (aHSCs) and studying them in standard tissue culture dishes (two-dimensional [2D]). In contrast, modeling of fibrosis in a biofabricated three-dimensional (3D) construct allows us to study changes to the environment, such as extracellular matrix (ECM) composition and structure, and tissue rigidity. In the current study, we used aHSCs produced through subcultures in 2D and encapsulated them in a 3D collagen gel to form spherical constructs. In parallel, and as a comparison, we used an established HSC line, LX-2, representing early and less severe fibrosis. Compared with LX-2 cells, the aHSCs created a stiffer environment and expressed higher levels of TIMP1 and LOXL2, all of which are indicative of advanced liver fibrosis. Collectively, this study presents a fibrosis model that could be incorporated with multi-cellular models to more accurately reflect the effects of a severe fibrotic environment on liver function.

Over 1 million burn injuries are treated annually in the United States, and current tissue engineered skin fails to meet the need for full-thickness replacement. Bioprinting technology has allowed fabrication of full-thickness skin and has demonstrated the ability to close full-thickness wounds. However, analysis of collagen remodeling in wounds treated with bioprinted skin has not been reported. The purpose of this study is to demonstrate the utility of bioprinted skin for epidermal barrier formation and normal collagen remodeling in full-thickness wounds. Human keratinocytes, melanocytes, fibroblasts, dermal microvascular endothelial cells, follicle dermal papilla cells, and adipocytes were suspended in fibrinogen bioink and bioprinted to form a tri-layer skin structure. Bioprinted skin was implanted onto 2.5 × 2.5 cm full-thickness excisional wounds on athymic mice, compared with wounds treated with hydrogel only or untreated wounds. Total wound closure, epithelialization, and contraction were quantified, and skin samples were harvested at 21 days for histology. Picrosirius red staining was used to quantify collagen fiber orientation, length, and width. Immunohistochemical (IHC) staining was performed to confirm epidermal barrier formation, dermal maturation, vascularity, and human cell integration. All bioprinted skin treated wounds closed by day 21, compared with open control wounds. Wound closure in bioprinted skin treated wounds was primarily due to epithelialization. In contrast, control hydrogel and untreated groups had sparse wound coverage and incomplete closure driven primarily by contraction. Picrosirius red staining confirmed a normal basket weave collagen organization in bioprinted skin-treated wounds compared with parallel collagen fibers in hydrogel only and untreated wounds. IHC staining at day 21 demonstrated the presence of human cells in the regenerated dermis, the formation of a stratified epidermis, dermal maturation, and blood vessel formation in bioprinted skin, none of which was present in control hydrogel treated wounds. Bioprinted skin accelerated full-thickness wound closure by promoting epidermal barrier formation, without increasing contraction. This healing process is associated with human cells from the bioprinted skin laying down a healthy, basket-weave collagen network. The remodeled skin is phenotypically similar to human skin and composed of a composite of graft and infiltrating host cells. IMPACT STATEMENT: We have demonstrated the ability of bioprinted skin to enhance closure of full-thickness wounds through epithelialization and normal collagen remodeling. To our knowledge, this article is the first to quantify collagen remodeling by bioprinted skin in full-thickness wounds. Our methods and results can be used to guide further investigation of collagen remodeling by tissue engineered skin products to improve ongoing and future bioprinting skin studies. Ultimately, our skin bioprinting technology could translate into a new treatment for full-thickness wounds in human patients with the ability to recapitulate normal collagen remodeling in full-thickness wounds.

Introduction Upon injury, skeletal muscle undergoes a multiphase process beginning with degeneration of the damaged tissue, which is accompanied by inflammation and finally regeneration. One consequence of an injured microenvironment is excessive production of reactive oxygen species, which results in attenuated regeneration and recovery of function ultimately leading to fibrosis and disability. The objective of this research was to test the potential of the antioxidant, N-Acetyl-L-Cysteine (NAC), as a mediator of reactive oxygen species damage that results from traumatic muscle injury in order to support repair and regeneration of wounded muscle tissue and improve function recovery. Materials and Methods Adult female Lewis rats were subjected to compartment syndrome injury as previously published by our group. Rats received intramuscular injections of NAC or vehicle at 24, 48, and 72 hours postinjury. Muscle function, tissue fibrosis, and the expression of myogenic and angiogenic markers were measured. Results Muscle function was significantly improved, and tissue fibrosis was significantly decreased in NAC-treated muscles. Conclusions These results suggest that NAC treatment of skeletal muscle after injury may be a viable option for the prevention of long-term fibrosis and scar formation, facilitating recovery of muscle function.

Lung cancer is the leading cause of cancer-related deaths worldwide, yet most currently used in vitro cancer models are confined to traditional 2D cell culture conditions. Recently; however, innovative 3D models such as tumor tissue equivalents (organoids) have been adopted by researchers to recapitulate tissue architecture and physiology in order to improve disease modeling and drug testing. We have hypothesized that 3D lung organoids, incorporating cells directly from patients, will enable personalized disease modeling and tumor cell characterization compared to traditional 2D cultures. Here, we discuss the fabrication of 3D lung cancer organoids using a rare cell source, pleural effusion aspirate. We tracked the phenotypic change that developed in short-term culturing and characterized the cell population within the organoids. We found that isolated patient cells embedded directly into organoids created anatomically relevant structures and displayed lung cancer specific behaviors compared to cultures that first grew in 2D conditions. Additionally, we compared responses of patient cells from pleural effusion aspirates to chemotherapy in 2D and 3D cell culture systems. Our results show that cells in 2D cultures are more sensitive to treatment when compared with 3D organoids. Collectively, we have been able to isolate tumor cells from pleural effusion fluid of lung cancer patients and create organoids that display in vivo like anatomy and drug response. This technology can serve as a more accurate disease model for studying tumor progression and drug development.

Pulsed electromagnetic field (PEMF) is emerging as innovative treatment for regulation of inflammation, which could have significant effects on tissue regeneration. PEMF modulates inflammatory processes through the regulation of pro- and anti-inflammatory cytokine secretion during different stages of inflammatory response. Consistent outcomes in studies involving animal and human tissue have shown promise for the use of PEMF as an alternative or complementary treatment to pharmaceutical therapies. Thus, PEMF treatment could provide a novel nonpharmaceutical means of modulating inflammation in injured tissues resulting in enhanced functional recovery. This review examines the effect of PEMF on immunomodulatory cells (e.g., mesenchymal stem/stromal cells [MSCs] and macrophages [M(Φ)]) to better understand the potential for PEMF therapy to modulate inflammatory signaling pathways and improve tissue regeneration. This review cites published data that support the use of PEMF to improve tissue regeneration. Our studies included herein confirm anti-inflammatory effects of PEMF on MSCs and M(Φ).

Malignant pleural effusion (MPE) is indicative of terminal malignancy with a uniformly fatal prognosis. Often, two distinct compartments of tumour microenvironment, the effusion and disseminated pleural tumours, co-exist in the pleural cavity, presenting a major challenge for therapeutic interventions and drug delivery. Clinical evidence suggests that MPE comprises abundant tumour-associated myeloid cells with the tumour-promoting phenotype, impairing antitumour immunity. Here we developed a liposomal nanoparticle loaded with cyclic dinucleotide (LNP-CDN) for targeted activation of stimulators of interferon genes signalling in macrophages and dendritic cells and showed that, on intrapleural administration, they induce drastic changes in the transcriptional landscape in MPE, mitigating the immune cold MPE in both effusion and pleural tumours. Moreover, combination immunotherapy with blockade of programmed death ligand 1 potently reduced MPE volume and inhibited tumour growth not only in the pleural cavity but also in the lung parenchyma, conferring significantly prolonged survival of MPE-bearing mice. Furthermore, the LNP-CDN-induced immunological effects were also observed with clinical MPE samples, suggesting the potential of intrapleural LNP-CDN for clinical MPE immunotherapy.

Current in vitro three-dimensional (3D) models of liver tissue have been limited by the inability to study the effects of specific extracellular matrix (ECM) components on cell phenotypes. This is in part due to limitations in the availability of chemical modifications appropriate for this purpose. For example, hyaluronic acid (HA), which is a natural ECM component within the liver, lacks key ECM motifs (e.g. arginine–glycine–aspartic acid (RGD) peptides) that support cell adhesion. However, the addition of maleimide (Mal) groups to HA could facilitate the conjugation of ECM biomimetic peptides with thiol-containing end groups. In this study, we characterized a new crosslinkable hydrogel (i.e. HA-Mal) that yielded a simplified ECM-mimicking microenvironment supportive of 3D liver cell culture. We then performed a series of experiments to assess the impact of physical and biochemical signaling in the form of RGD peptide incorporation and transforming growth factor ß (TGF-ß) supplementation, respectively, on hepatic functionality. Hepatic stellate cells (i.e. LX-2) exhibited increased cell–matrix interactions in the form of cell spreading and elongation within HA-Mal matrices containing RGD peptides, enabling physical adhesions, whereas hepatocyte-like cells (HepG2) had reduced albumin and urea production. We further exposed the encapsulated cells to soluble TGF-ß to elicit a fibrosis-like state. In the presence of TGF-ß biochemical signals, LX-2 cells became activated and HepG2 functionality significantly decreased in both RGD-containing and RGD-free hydrogels. Altogether, in this study we have developed a hydrogel biomaterial platform that allows for discrete manipulation of specific ECM motifs within the hydrogel to better understand the roles of cell–matrix interactions on cell phenotype and overall liver functionality.

The field of three-dimensional (3D) bioprinting has advanced rapidly in recent years. Significant reduction in the costs associated with obtaining functional 3D bioprinting hardware platforms is both a cause and a result of these advances. As such, there are more laboratories than ever integrating bioprinting methodologies into their research. However, there is a lack of standards in the field of biofabrication governing any requirements or characteristics to support cross-compatibility with biomaterial bioinks, hardware, and different tissue types. Here we describe a modular extracellular matrix (ECM) inspired bioink comprised of collagen and hyaluronic acid base components that: 1) employ reversible internal hydrogen bonding forces to generate thixotropic materials that dynamically reduce their elastic moduli in response to increased shear stress, thus enabling increased compatibility with printing hardware; and 2) modular addons in the form of chemically-modified fibronectin and laminin that when covalently bound within the bioink support a variety of tissue types, including liver, neural, muscle, pancreatic islet, and adipose tissue. These features aim to accelerate the deployment of such bioinks for tissue engineering of functional constructs in the hands of various end users.

Three-dimensional (3D) cell culture systems, such as tumor organoids and multicellular tumor spheroids, have been developed in part as a result of major advances in tissue engineering and biofabrication techniques. 3D cell culture offers great capabilities in drug development, screening, testing, and precision medicine owing to its physiological accuracy. However, since the inception of 3D systems, few methods have been reported to successfully analyze cell viability quantitatively within hydrogel constructs. In this study, we describe and compare commercially available viability assays developed for two-dimensional (2D) applications for use in 3D constructs composed of organic, synthetic, or hybrid hydrogel formulations. We utilized Promega's CellTiter-Glo(®), CellTiter-Glo 3D, and CellTiter 96(®) MTS Assay along with Thermo Fisher's PrestoBlue(™) assay to determine if these assays can be used accurately in 3D systems. Compared with direct cell viability commonly used in 2D cell culture, our results show cellular health output inaccuracies among each assay in differing hydrogel formulations. Our results should inform researchers of potential errors when using cell viability measurements in 3D cultures and conclude that microscopic imaging should be used, in combination, for validation. IMPACT STATEMENT: Three-dimensional (3D) tissue organoids models are a valuable tool not only for studying drug toxicity but also for understanding human embryonic development, intra-tissue morphogenesis, and mechanisms of disease. In cancer organoids, such 3D models may be used for preclinical chemotherapy screening and for understanding cell death and viability mechanisms under physiologically relevant conditions. Cell viability assays are necessary for assessing the effect of biological reagents on cellular health and have been used on in vitro cell cultures for many years. With the increase of 3D systems in cellular biology research to determine therapeutic efficacy, two-dimensional assays that measure cell viability are being used outside their intended use on 3D constructs. In this study, we assess the accuracy of using various commercially available cell viability assays on different 3D hydrogel constructs to help researchers understand expected variability in their experimentation along microscopic imaging validation.

There is a need for effective wound treatments that retain the bioactivity of a cellular treatment, but without the high costs and complexities associated with manufacturing, storing, and applying living biological products. Previously, we developed an amnion membrane-derived hydrogel and evaluated its wound healing properties using a mouse wound model. In this study, we used a full thickness porcine skin wound model to evaluate the wound-healing efficacy of the amnion hydrogel and a less-processed amnion product comprising a lyophilized amnion membrane powder. These products were compared with commercially available amnion and nonamnion wound healing products. We found that the amnion hydrogel and amnion powder treatments demonstrated significant and rapid wound healing, driven primarily by new epithelialization versus closure by contraction. Histological analysis demonstrated that these treatments promote the formation of a mature epidermis and dermis with similar composition to healthy skin. The positive skin regenerative outcomes using amnion hydrogel and amnion powder treatments in a large animal model further demonstrate their potential translational value for human wound treatments. Significance statement This study demonstrates the efficacy of amnion hydrogel and amnion powder wound healing products in a large animal model. This further demonstrates their potential translational value for human wound treatments.

Introduction: Peritoneal carcinomatosis is a late-stage complication of appendiceal cancer, leading to decreased life expectancy and poor quality of life. Cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) has improved overall survival. Despite this advancement, there remain challenges in determining which patients may benefit from the procedure. Hyperthermia has been shown to potentiate chemotherapy toxicity through interfering with DNA repair, suggesting DNA repair proteins could serve as a predictive marker of clinical outcomes. Herein we applied patient derived tumor organoids (PTOs) to examine the personalized impact of DNA repair mechanisms in efficacy of HIPEC. Methods: Tumor specimens were obtained from patients undergoing CRS under IRB protocols. Specimens were dissociated and incorporated into a collagen-hyaluronic acid organoid model and treated with clinical HIPEC regimens using mitomycin C (MMC) and oxaliplatin under normothermic (37°C) and heated conditions (42°C) for two hours. The post-treatment viability average responses were aggregated and used for statistical analysis. Treated organoids and tissues were processed histologically and qPCR was performed on patient tumor cells to determine expression of DNA damage markers and DNA damage response. Results: From June 2019-October 2021, 59 appendiceal tumor tissues from 35 patients with peritoneal carcinomatosis were acquired. 53 PTO groups, from 17 high grade and 36 low grade tumors, were successfully fabricated and tested for HIPEC sensitivity. Hyperthermia potentiated the cytotoxic effect for both MMC (35% vs 57%, p Conclusions: Decreased expression of PTIP offers resistance in appendiceal cancer patients treated with HIPEC. PTOs can serve to determine personalized efficacy of HIPEC regimens both at the level of selection of perfusion chemotherapy agent or application of hyperthermia. Citation Format: Steven D. Forsythe, Richard A. Erali, Preston Laney, Allan M. Johansen, Shyama Sasikumar, Perry Shen, Edward A. Levine, Shay Soker, Konstantinos I. Votanopoulos. Low PTIP expression is linked to HIPEC resistance in appendiceal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3076.

Bladder cancer has high recurrence rates, which leads to treatment difficulties and reduced survival. Field cancerization is the prevailing idea for why bladder cancers recur with high frequency, and it centers around genetic and epigenetic changes in tissue that lead to conditions favoring recurrence. However, the specifics of these alterations are not well understood or described. The tumor microenvironment (TME) has been implicated as a strong proponent of oncogenesis in many organ systems, including the bladder. The TME comprises stromal cells, paracrine factors, and extracellular matrix (ECM) components, which may contribute to field cancerization. As such, identifying the hallmarks of these alterations may expedite the prognosis of recurrence. For this purpose, we fabricated bladder cancer organoids of varied cancer grades, with which we developed a texture-based grading system. Image texture is characterized by filtering images and finding their similarity. The similar images are clustered, and the cumulative histogram of clusters is formed to find the closest training image. In two independent image data sets of 54 and 76 images, respectively, with different imaging protocols, 100% and 92% accuracy were achieved.

The therapeutic efficacy of clinically applied mesenchymal stromal cells (MSCs) is limited due to their injection into harsh in vivo environments, resulting in the significant loss of their secretory function upon transplantation. A potential strategy for preserving their full therapeutic potential is encapsulation of MSCs in a specialized protective microenvironment, for example hydrogels. However, commonly used injectable hydrogels for cell delivery fail to provide the bio-instructive cues needed to sustain and stimulate cellular therapeutic functions. Here we introduce a customizable collagen I-hyaluronic acid (COL-HA)-based hydrogel platform for the encapsulation of MSCs. Cells encapsulated within COL-HA showed a significant expansion of their secretory profile compared to MSCs cultured in standard (2D) cell culture dishes or encapsulated in other hydrogels. Functionalization of the COL-HA backbone with thiol-modified glycoproteins such as laminin led to further changes in the paracrine profile of MSCs. In depth profiling of more than 250 proteins revealed an expanded secretion profile of proangiogenic, neuroprotective and immunomodulatory paracrine factors in COL-HA-encapsulated MSCs with a predicted augmented pro-angiogenic potential. This was confirmed by increased capillary network formation of endothelial cells stimulated by conditioned media from COL-HA-encapsulated MSCs. Our findings suggest that encapsulation of therapeutic cells in a protective COL-HA hydrogel layer provides the necessary bio-instructive cues to maintain and direct their therapeutic potential. Our customizable hydrogel combines bioactivity and clinically applicable properties such as injectability, on-demand polymerization and tissue-specific elasticity, all features that will support and improve the ability to successfully deliver functional MSCs into patients.