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.

Translational Relevance

Clinical trial accrual in rare diseases is limited by low incidence and lack of research models. Herein, we apply patient-derived tumor organoids, enhanced with autologous immune system, to study the efficacy of checkpoint inhibitors in appendiceal cancer. Appendiceal cancer is an orphan primary, with limited research models, and historically resistant to systemic chemotherapy. We explored the concept of using appendiceal cancer immunocompetent organoids as a preclinical companion platform, for the ex vivo study of the interaction between tumor and host's immune system, possibly optimizing selection of clinical trial candidates, at the level of the individual patient.

Appendiceal cancer is a rare tumor with an estimated incidence of 0.97 per 100,000 people in the United States (1). Peritoneal dissemination in appendiceal cancer increases treatment complexity and makes control of disease inherently more challenging. Surgical management of diffuse peritoneal involvement relies on cytoreductive surgery (CRS) with heated intraperitoneal chemotherapy (HIPEC; ref. 2).

The utility of systemic chemotherapy for low-grade appendiceal (LGA) neoplasms has been debated and there are few studies examining this outcome (3). Patient tumor organoids (PTO) have been demonstrated as reliable ex vivo models to study a variety of cancers such as appendiceal cancer, peritoneal mesothelioma, and colorectal cancer with peritoneal metastases (4–7). Application of PTOs in metastatic colorectal and gastroesophageal cancer enrolled in phase I/II clinical trials, recapitulated patient response to chemotherapy with an 88% positive predictive value and 100% negative predictive value (8).

Immunotherapy is a rapidly growing area of research in cancer drug development with promising results for a variety of cancers including colorectal, esophageal, and melanoma (9–11). Since 2017, checkpoint inhibitors (CPI), have gained widened FDA approval for treatment of solid tumors in patients with high tumor mutational burden (TMB-H) and patients whose tumors demonstrate mismatch repair deficiency, or phenotypic evidence of microsatellite instability (MSI-H; refs. 12–14). Despite broader FDA approval, the rarity of appendiceal cancer makes accrual in immunotherapy clinical trials exceedingly difficult. Immunotherapy efficacy data in appendiceal cancer are currently limited to a single case report (15). In addition, preclinical platforms such as patient-derived xenograft (PDX) models and cell lines either do not exist for the majority of rare diseases or are associated with a timeframe of deployment that is not aligned with the clinical needs of the patient.

Herein, we utilized PTOs as a platform to study the efficacy of immunotherapy in appendiceal cancer organoids from patients presenting with peritoneal dissemination. We hypothesized that appendiceal cancer PTOs can be reproducibly applied to generate preclinical immunotherapy efficacy data, with the potential to broaden drug indications, while defining a focused personalized approach in clinical trial design in an orphan primary.

Tissue and whole-blood specimens were obtained from 26 patients with appendiceal cancer with peritoneal dissemination who underwent CRS/HIPEC procedures between September 2019 and May 2021. Specimens were obtained in accordance to Wake Forest Baptist Medical Center guidelines and under an institution-approved Institutional Review Board (IRB) protocol. Specimens were placed in RPMI media and transferred to the Wake Forest Organoid Research Center (WFORCE) for processing within a 2-hour targeted framework from surgical resection.

Tumor procurement and processing

Once specimens were received in the laboratory, tumors were washed in PBS with 100 U/mL penicillin-streptomycin, 5 μg/mL gentamicin, and 5 μg/mL amphotericin B for two 5-minute cycles. A portion of each specimen was saved for whole-tissue histology. The remaining specimen portions were minced finely and placed in a 15 mL conical in a 3 mL solution of DMEM with 100,000 cytidine deaminase (CDA) units per mL collagenase HA (001–1050; VitaCyte), 22,000 narcissus pseudonarcissus agglutinin (NPA) units per mL protease (003–1000; VitaCyte), and 50 mmol/L n-acetyl l-cysteine (A9165; Sigma-Aldrich) per gram of tissue for up to 120 minutes under agitation at 37°C. Upon complete tissue dissolution, enzymatic digestion was terminated with 5 mL cold DMEM-10. The resultant tumor solution was filtered through a 100-μm pore-size vacuum filtration kit (SCNY00100; Millipore Sigma) and centrifuged to isolate a cell pellet. Supernatant was removed and the cell pellet resuspended with Red Blood Cell Lysis Buffer (Abcam) according to company protocol. Lysis buffer was discarded, and the cell pellet resuspended and counted using a NucleoCounter NC-200 (Chemometec). Whole blood was obtained from patients for processing and retrieval of immunocompetent cells using Ficoll-Paque PLUS and corresponding protocol (GE Healthcare). Normal lymph nodes from 2 patients and normal spleen tissue from one patient were obtained for additional comparative analysis with blood-derived immunocompetent cells. Lymph nodes and spleen were processed similarly to whole tissue as described above.

Organoid fabrication and culture

The tumor cell pellet was resuspended with the thiol-modified hyaluronan/heparin (Heprasil; Advanced Biomatrix) and methacrylated collagen (PhotoCol; Advanced Biomatrix) solution in a 1:3 volume ratio at a cell density of 10 million cells per mL. PTOs were then created by seeding 5 μL of the hydrogel/cell mixture into individual wells of a 96-well nontissue culture–treated plate and then photocrosslinked by exposure to ultraviolet light (365 nm, 18W/cm2) from a BlueWave 75 V.2 UV spot lamp (Dymax Corp.) for 2 seconds to form crosslinking via thiol-modified hyaluronan/heparin and methacrylated collagen. PTOs were cultured in 200 μL media containing DMEM-F12 with 5% FBS, 1% penicillin–streptomycin, 1% l-glutamine, 50 ng/mL EGF (PHG 0313; Thermo Fisher Scientific), and 10 μmol/L Y-27632 (S1049) with media changes after 3 to 4 days.

Immune-enhanced PTOs (iPTO) were created by combining the immunocompetent cells from each patient's corresponding whole blood (blood iPTO) and spleen or nodal lymph tissue (lymph iPTO) in a ratio ranging from 1:1 to 1:10 according to cell yield with tumor cells and seeded onto plates as described above. The organoids in addition to tumor and CD8+ cells, contain CD4+ and antigen presenting cells (APC), as well as stroma as described previously (4, 6, 7). Organoids were cultured for 7 days prior to treatment.

Drug screens

Organoids were subsequently treated after 7 days of culture with 100 nmol/L of pembrolizumab (A2002, Selleckchem), ipilimumab (A2001, Selleckchem), or nivolumab (A2005, Selleckchem). This drug concentration corresponds to dosing recommendations for a patient weighing 70 kgs. Media was aspirated from the wells and drug solutions mixed in culture media were added to each well individually. Organoids remained in drug-containing media solution for 3 days prior to endpoint viability assessment.

Organoid viability assessment

After 3 days of incubation in drug-containing media, organoids were assessed with LIVE/DEAD staining and CellTiter-Glo 3D viability assays. LIVE/DEAD staining (L3224; Invitrogen) was performed according to the manufacturer's protocol and incubated at 37°C for 2 hours prior to imaging. Fluorescent imaging was performed on whole organoids using a Leica TCS LSI macro confocal microscope (Leica Microsystems Inc.). Images from red and green channels were overlaid and stacked in maximum projection.

Quantitative viability was assessed utilizing CellTiter-Glo 3D Cell Viability Assay (G968B; Promega). Half the media (100 μL) was removed from individual wells and 100 μL of ATP assay was added to each well, and incubated at room temperature on a shaker for 30 minutes. Well contents were transferred to a Costar White Polystyrene 96-well Assay Plate (3912) and analyzed with a Veritas Microplate Luminometer (Turner BioSystems).

Organoid tissue characterization

Organoids were fixed for histology on days 1 and 10 of culture in 4% paraformaldehyde (PFA) for 4 hours. Organoids were processed, paraffin embedded, and sectioned at 5-μm intervals for staining. Organoid sections were stained on glass microscope slides with hematoxylin and eosin (H&E).

Additional staining was performed with IHC to characterize programmed death ligand-1 (PD-L1), cluster of differentiation 8 (CD-8), cytokeratin 20 (CK-20), and granzyme B biomarker expression. Unstained slides underwent antigen retrieval in a pH 6 citrate buffer solution prior to blocking with Dako Protein Block for 30 minutes. Fluorescent IHC was performed by applying primary antibodies PD-L1 (ab205921, Abcam, rabbit), CD-8 (ab4055, Abcam, rabbit), CK-20 (MA5–13263, Invitrogen, mouse), granzyme B (ab4059, Abcam, rabbit), and cleaved caspase 3 (9661S, Cell Signaling Technology, rabbit) to slides in ratios of 1:500, 1:200, 1:200, 1:100, and 1:400 in Dako Antibody Diluent, respectively. After incubation for 1 hour, appropriate species reactive secondary to Alexa Fluor 488 or Alexa Fluor 594 antibodies (Biotium) were applied to samples for 1 hour at a 1:1,000 dilution. Sections were then incubated with DAPI for 5 minutes prior to finalization with coverslipping. An Olympus BX-63 upright fluorescent microscope was used to image the sections.

Cell membrane tracking

For patient 21, prior to organoid encapsulation, unsorted tumor cells and immune cells were tagged using DIO (tumor cells) and DII (immune cells) fluorescent dyes (Thermo Fisher Scientific) according to company protocol. Organoids were cultured, treated, and imaged as described above. Fluorescent images were analyzed using ImageJ Fiji analysis software using red and green pixel analysis. The percentage ratio of total green to red pixel counts was obtained and tumor cell images were converted from green to yellow.

Definition of treatment response

Immunotherapy efficacy in organoids is currently undefined. Herein, we developed a conservative approach for considering an organoid to be responsive to immunotherapy, consisting of three distinct criteria that simultaneously must be met by iPTOs: (i) demonstrate a statistically significant reduction in cell viability when compared with iPTO untreated (control) organoids (e.g., iPTO control vs. iPTO treated); (ii) demonstrate a statistically significant reduction in cell viability when comparing treated organoids from immune-enhanced with the nonimmune-enhanced counter conditions (e.g., pembrolizumab-treated iPTO vs. pembrolizumab-treated PTO); and (iii) exhibit a post-immunotherapy ATP viability greater than 50%.

The lower threshold of immunotherapy efficacy in organoids is unknown. Herein we arbitrarily selected 50% killing of the tumor as the lowest threshold suggestive of immunotherapy response. This number can be increased or decreased based on the desired tumor response in need to be studied or the kinetics and the tumor biology of every individual patient, demonstrating the plasticity of the platform.

Statistical analysis

All data is expressed as mean ± SD for each experimental group. Each treatment and condition combination consisted of three or more organoids for analysis. ATP assay values of treated organoids were standardized to condition-matched (iPTO or PTO) controls prior to statistical analysis. Upon review of the CellTiter-Glo results, outlier ATP replicate values were removed by a committee of researchers to improve the rigor of the final analysis. Two-sample t tests were also used to assess whether cell viability values were different between immune-enhanced and nonimmune-enhanced counter conditions. We intentionally chose a rigorous threshold for determining whether a particular organoid showed a response to immunotherapy. This threshold requires all three treatment response conditions (identified in the previous section) be met in order to consider an organoid as being a treatment response. This approach was used to reduce the probability of a type I error occurring. Specifically, the chance that the two t tests described above would both be significant at P < 0.05 (rather than because both indicated evidence of a treatment response) would be 0.25%. Furthermore, if the probability that post immunotherapy ATP viability is less than 50% were a random event (i.e., 50% chance that it would occur by chance) then the combined probability that all three events would occur simultaneously by chance would be 0.125% or 12.5 in 10,000. Drug screen studies were determined to be successful for a patient if untreated control PTOs demonstrated adequate viability at day 10 of culture, which coincided with termination of drug screens, and each condition had a counter control condition with adequate viability. Adequate viability is described as blank value of less than 1% of control condition. Statistical analysis was performed with GraphPad Prism (GraphPad Software Inc.) and a P value of more than 0.05 was used as the threshold for statistical significance. Reported P values indicate significance to parts 1 and 2 of the treatment response definitions outlined above.

Patient characteristics

A total of 26 patients with appendiceal neoplasms were enrolled in the study, including 16 of 26 (61.5%) LGA and 10 of 26 (38.5%) HGA primaries (Table 1). Thirteen patients (50%) had prior systemic chemotherapy, while 8 patients (30.8%) had no prior surgical or medical treatment. Genetic analysis was reported for 9 patients with FoundationOne sequencing panels (16) and for 1 patient with STRATA. Both sequencing panels report PD-L1 expression by the Tumor Proportion Score (TPS; Table 1). Similarly, microsatellite instability and mismatch repair (MMR) testing were clinically performed on 3 patients, who were determined to be MMR proficient. Patient demographic information, including race and ethnicity, are not reported to minimize the potential for patient identification given the rarity of this disease.

Organoid characteristics and biofabrication timeline

All specimens underwent initiation of an hour-long enzymatic digestion with collagenase, n-acetylcysteine, and protease within 2.5 hours from tissue procurement (Fig. 1). Cell counts were completed within an additional hour post digestion followed by stabilization of tumor cells in supportive extracellular matrix (ECM). The above sequence resulted in fabrication of immune system–enhanced organoids with a time frame of less than 5 hours post specimen resection. iPTOs were enhanced with white blood cells (WBC) only in 23 of 26 (88.5%) cases, while normal spleen tissue only was used in 1 of 26 (3.8%) patients. Two patients (7.7%) had iPTOs made with both WBC and lymph nodes as separate comparative groups. PTOs without immune components were generated for comparative controls for all 26 tumor specimens.

Organoid characterization

Fluorescent IHC was performed to characterize tumor cell and leukocyte interactions. Abundance of CK20-positive epithelial cells and CD8+ T cells were observed on day 1, indicating viable appendiceal tumor cells and immune cells cocultured within iPTOs (Fig. 2A). The expression of PD-L1 was confirmed on CK20-positive appendiceal tumor cells along with no discernable granzyme B staining, indicating lack of CD8 T-cell–mediated cytotoxic activity in the absence of CPIs (Fig. 2A and B). At day 10, iPTOs responding to immunotherapy demonstrated increased granzyme B expression (red) and cleaved caspase 3 (red) with corresponding decrease in CK20 appendiceal tumor cells (green; Fig. 2C).

Viability of PTOs and iPTOs was also qualitatively evaluated with LIVE/DEAD staining on days 1 and 10 demonstrating stable CK20 cellular populations in the absence of immunotherapy response (Fig. 3). Cell membrane tagging, performed for patient 21, demonstrated the presence of immune and tumor cell populations throughout the course of the study (Fig. 4). In pembrolizumab-treated organoids, tagged tumor cell populations demonstrated a decrease in the ratio of tumor cells to immune cells, as a result of CPI application (25.1% vs. 38.6% of total cells; Fig. 4). In addition to the decrease in the proportion of tumor cells within the organoid, there was also a decrease in the organoid size itself when compared with day 1, suggesting ECM remodeling by the imported cellular lymph node component.

iPTO response to immunotherapy

After 7 days of culture, 72-hour long drug screens were performed on appendiceal PTOs and iPTOs with pembrolizumab, ipilimumab, or nivolumab. CD8+ T cells were 96.3% viable at 1 week in coculture with tumor cells as demonstrated by flow cytometry (Supplementary Fig. S1). Drug screens were deemed successful if the control condition for PTO and iPTO demonstrated adequate ATP viability after 10 days of culture. Successful drug screen studies were conducted in 19 of 26 (73.1%) patients, specifically 6 of 10 (60%) in HGA patients and 13 of 16 (81.3%) in LGA patients (Table 1).

Six patients (6/19, 31.6%) demonstrated iPTO response to immunotherapy that was defined as at least 50% cellular death posttreatment for the purposes of the study (Table 1). Overall, response was observed in 3 of 6 (50%) HGA and 3 of 13 (23.1%) LGA iPTOs. Pembrolizumab was effective in 4 of 19 (21.1%) iPTOs with an average post-treatment viability of 22.9% (Fig. 5A). Nivolumab was effective in 2/19 (10.5%) iPTOs, demonstrating an average posttreatment viability of 27.7% (Fig. 5B). Ipilimumab was not found to be effective in any of the examined specimens. A comparative set of blood iPTOs and lymph iPTOs were fabricated for 1 patient (patient 4, HGA) for which pembrolizumab was equally effective (5.3% vs. 5.8% post-treatment viability, respectively; Fig. 3B). We have noticed an increased cell viability in organoids made from patient 2 (Fig. 3A) and treated with CPI. Although it is possible anti–PD-1 antibodies may bind to cell surface receptors (PD-1 or other) that trigger signaling pathways leading to enhanced metabolic activity, or there is a transient increased metabolism response to cytotoxic conditions, this does not likely represent in vivo response conditions.

Organoid correlation with patient clinical response

At this time, 2 patients (patients 2 & 17) have received immunotherapy in the clinical setting and were both correlating with the organoid response. The first patient was administered pembrolizumab due to PD-L1 expression. The patient did not respond to immunotherapy, which correlated with lack of response of their organoids. The second patient underwent pembrolizumab treatment for a TMB > 10 without radiologic response or clinical response, similar to the corresponding appendiceal organoids.

The rarity of appendiceal cancer is a major limitation for the identification of new therapies through recruitment in clinical trials. We have previously shown the versatility of PTOs in studying the personalized efficacy of systemic as well as intraperitoneal chemotherapy in a variety of rare cancers, including appendiceal neoplasms, sarcomas, and peritoneal mesothelioma (5, 6).

Immunotherapy is an approved therapy for solid tumors with microsatellite instability and increased mutational burden, based on the assumption that increased mutational load will result in increased tumor antigenicity and therefore activation of adaptive immunity mechanisms. It is well documented that not all patients with MSI or increased TMB will respond to immunotherapy, while it is highly unlikely that these two indications are the only scenarios where immunotherapy will be beneficial to a cancer patient. In addition, the cost of immunotherapy treatment is not insignificant to be applied based on generalized indications. Therefore, there is an unmet need for an approach to explore the possibility of ex vivo identification of tumors that will respond to immunotherapy at the level of the individual patient and outside the framework of current sequencing indications. The aim of this study is to demonstrate feasibility of an iPTO platform in generating preclinical immunotherapy efficacy data in a rare primary such as appendiceal cancer where level I clinical data are lacking.

Organoids recapitulate the tumor microenvironment by incorporating not only tumor cells but also cells from tumor-associated stroma. Immunotherapy works through activation of a patient's own immune system, therefore adding a patient-matched immune system within the organoid is necessary to test CPI efficacy. Herein, PTOs have been cocultured with patient-matched spleen, lymph nodes, and/or peripheral blood mononuclear cells (PBMC) from peripheral blood. The power in the current study is not sufficient to identify variations in construct behavior, but from our earlier experience with melanoma immunocompetent organoids, addition of PBMCs suffices when the objective of the enrichment is limited to evaluation of immunotherapy response (4). In addition, it simplifies the logistics of coculturing, without the need of additional tissue processing.

The relationship between PD-L1 and response to immunotherapy is unclear, with the majority of data not supporting a reliably predictive role in other primaries such gastric and gastroesophageal junction cancers (17). Literature review at the time of this manuscript revealed a single case report of immunotherapy efficacy in an appendiceal cancer patient that did not express PD-L1 and was microsatellite stable (15). Similarly, we identified a patient with no PD-L1 expression that exhibited significant treatment efficacy to pembrolizumab, as well as patients who did not respond to any immunotherapy drug despite PD-L1 ≥ 1%. The organoid observed response rate in our cohort is higher than what would be expected from non–MSI-H gastrointestinal (GI) malignancies, but it is likely the two groups are not comparable. The group of patients with peritoneal dissemination have significant volume of disease presenting with multiple, often more than 30 to 50 distinct peritoneal lesions and a multiclonal makeup that directly increases antigenicity. We believe that it is this antigenicity that drives the observed immune responses and not the PD-L1 status, TMB-H, or MSI that in appendiceal cancer is determined to be very low at 2.8%, 2.2%, and 2.2%, respectively (18). The above demonstrates the pitfalls of using isolating sequencing results in treatment decisions, that with few exceptions, cannot take into consideration interactions between variable existing genomic pathways within the entire tumor.

Interestingly, similarly to our prior melanoma work (4), we observed differential treatment efficacies for pembrolizumab and nivolumab in iPTOs derived from the same patient. While studies currently support the clinical interchangeability of these two agents (19), it seems at least at an iPTO level, the efficacy of these two drugs was not equal for the same patient. It is unknown if this can be explained by structural property differences such as binding affinities for PD-1 receptors or epitope binding within the PD-1 loop that is dominated by interactions with the N segment for nivolumab and CD segment for pembrolizumab (20).

We have previously shown lack of response to chemotherapy for organoids derived from low-grade appendiceal primaries (5). However, though often indolent, low-grade appendiceal cancer carries significant morbidity in patients with a volume of disease that is not amenable to CRS/HIPEC or has recurred after prior multiple cytoreductions. The efficacy of immunotherapy for 23.1% of LGA patients (3/13) in this study could open opportunities for new applications of CPIs. Currently, there is only a single US-based Phase II clinical trial examining responses to ipilimumab and nivolumab for the treatment of mucinous appendiceal and colorectal tumors (21). Interestingly, we did not identify appendiceal cancer iPTOs that responded to ipilimumab within the examined specimens. PTOs have shown tremendous value and promise in ex vivo characterization of a variety of malignant GI tumors and correlation with clinical response (8). The potential to deliver personalized results for each tumor can spare patients from harmful side effects of treatments for which they will obtain no benefit.

Limitations of the study include the modest power as well as the fact that immunotherapy is not currently considered as a routine treatment option in patients with appendiceal cancer with peritoneal dissemination. Therefore, correlation data between the organoid response and the clinical response of the corresponding patient to CPI treatment cannot be generated as we have previously demonstrated for melanoma iPTOs where the iPTO response to immunotherapy was similar to specimen clinical response in 85% (6/7) of patients (4). In addition, we remove mucin during the processing stage to preserve organoid integrity. Thus, our model does not account for the possibility of excessive mucin interference on drug delivery. Nevertheless, this the only available preclinical study on immunotherapy efficacy on appendiceal cancer primaries, suggesting that CPIs may be a promising option in a subpopulation of patients with appendiceal cancer that can be identified through development of iPTOs within 2 weeks from tissue procurement. Current enrollment in clinical trials is predominantly guided by tumor type with limited armamentarium in incorporating information relevant to cohort heterogeneity. The implications of a platform that can generate preclinical data for enrollment of the most suitable patients to the appropriate therapeutic schemas and agents may have a significant impact on understanding and management of rare diseases. Only upon validation from clinical studies, these methods may be potentially useful in personalized application of immunotherapy or possibly target selection of optimal candidates for clinical trials.

In conclusion, immunotherapy exhibits unexpected cytotoxic efficacy in a subset of appendiceal cancer immune-enhanced organoids from both low- and high-grade primaries possibly offering an opportunity for a more targeted approach in clinical trial design. Organoid technology could potentially identify immunotherapy responders at the level of the individual patient outside the current indications of microsatellite instability and increased TMB.

S. Soker reports a patent for Tumor Organoids pending. K.I. Votanopoulos reports grants from NORD and NCI R01CA249087 during the conduct of the study; in addition, K.I. Votanopoulos has a patent for Cancer Modeling Platforms and Methods Using the Same issued and a patent for Organoids Related to Immunotherapy and Methods of Preparing and Using the Same issued. No disclosures were reported by the other authors.

S.D. Forsythe: Data curation, formal analysis, validation, investigation, methodology, writing–original draft, writing–review and editing. R.A. Erali: Data curation, formal analysis, validation, investigation, methodology, writing–original draft, writing–review and editing. S. Sasikumar: Data curation, formal analysis, validation, methodology. P. Laney: Data curation, formal analysis, validation, methodology. E. Shelkey: Data curation, validation, methodology. R. D'Agostino Jr: Formal analysis, validation, methodology, writing–review and editing. L.D. Miller: Conceptualization, resources, project administration. P. Shen: Resources. E.A. Levine: Resources. S. Soker: Conceptualization, supervision, funding acquisition, writing–review and editing. K.I. Votanopoulos: Conceptualization, resources, supervision, funding acquisition, validation, investigation, writing–original draft, project administration, writing–review and editing.

We graciously acknowledge the work of Libby McWilliams and Kathleen Perry for their assistance in tissue procurement and data management. The authors would also like to acknowledge their funding for the project which was supported by the Wake Forest Dean's Hero Award, the Appendix Cancer Pseudomyxoma Peritonei Foundation (ACPMP), the National Organization of Rare Diseases (NORD), and NIH R01CA249087. S.D. Forsythe is supported by a grant from the NIH (T32CA247819). This work was supported by Wake Forest Tumor Tissue and Pathology Shared Resource NCI CCSG P30CA012197, National Organization of Rare Diseases (NORD), The Appendix Cancer Pseudomyxoma Peritonei Research Foundation (ACPMP), and NIH, T32CA247819 and NIH R01CA249087.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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