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Summary: Herein, we present a protocol for culturing patient-derived organoids (PDOs) of cervical cancer that includes workflows for tumor biopsy/resection tissue and cytobrush-sampled cells. We describe steps for PDO culture initiation, including rinsing, gentle dissociation, Lymphoprep separation, and cell assessment, as well as seeding cells from surgical and cytobrush tissue digestion. We then provide guidance on PDO maintenance and passage and techniques for producing conditioned medium. Overall, this protocol serves as a valuable guide for establishing and maintaining cervical cancer PDOs.For complete details on the use and execution of this protocol, please refer to Colbert et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Purpose: The immune system’s role in mediating the cytotoxic effects of chemoradiotherapy remains not completely understood. The integration of immunotherapies into treatment will require insight into features and timing of the immune microenvironment associated with treatment response. Here, we investigated the role of circulating neutrophils and tumor-associated myeloid cells (TSAMs) as potential agents and biomarkers for disease-related outcomes in locally advanced cervical cancer (LACC). Material and Methods: Hematologic parameters for two LACC patient cohorts, a retrospective clinical and a prospective translational cohort, were obtained at baseline, weekly during chemoradiotherapy for the retrospective cohort, biweekly during chemoradiotherapy for the prospective cohort, and at the first follow-up visit for both cohorts (mean 14.7 weeks, range 8.1–25.1 weeks for the prospective cohort and 5.3 weeks with a range of 2.7–9.0 weeks for the retrospective cohort). In both cohorts, baseline as well as mean and lowest on-treatment values for platelets, hemoglobin, absolute neutrophil count (ANC), and absolute lymphocyte count (ALC) were analyzed for correlations with disease-related outcomes. In the prospective cohort, circulating myeloid cells were isolated from peripheral blood mononuclear cells (PBMCs), and TSAMs were isolated from tumor tissue via a novel serial cytobrush sampling assay. The samples were analyzed by flow cytometry. Results: In both cohorts, the only hematologic parameter significantly associated with survival was elevated on-treatment mean ANC (mANC), which was associated with lower local failure-free and overall survival rates in the retrospective and prospective cohorts, respectively. mANC was not associated with a difference in distant metastases. CD11b+CD11c- TSAMs, which act as a surrogate marker for intratumoral neutrophils, steadily decreased during the course of chemoRT and nadier’d at week 5 of treatment. Conversely, circulating myeloid cells identified from PBMCs steadily increased through week 5 of treatment. Regression analysis confirmed an inverse relationship between circulating myeloid cells and TSAMs at this time point. Conclusions: These findings identify on-treatment mean neutrophil count as a predictor of disease-related outcomes, suggest that neutrophils contribute to chemoradiation treatment resistance, and demonstrate the importance of techniques to measure intratumoral immune activity.

Purpose: There are limited treatment options for locally advanced, unresectable pancreatic cancer (LAPC) and no likelihood of cure without surgery. Radiation offers an option for local control, but radiation dose has previously been limited by nearby bowel toxicity. Advances in on-board imaging and treatment planning may allow for dose escalation not previously feasible and improve local control. In preparation for development of clinical trials of dose escalation in LAPC, we undertook a dosimetric study to determine the maximum possible dose escalation while maintaining known normal tissue constraints. Methods and Materials: Twenty patients treated at our institution with either SBRT or dose-escalated hypofractionated IMRT (DE-IMRT) were re-planned using dose escalated SBRT to 70 Gy in 5 fractions to the GTV and 40 Gy in 5 fractions to the PTV. Standard accepted organ at risk (OAR) constraints were used for planning. Descriptive statistics were generated for homogeneity, conformality, OAR's and GTV/PTV. Results: Mean iGTV coverage by 50 Gy was 91% (±0.07%), by 60 Gy was 61.3% (±0.08%) and by 70 Gy was 24.4% (±0.05%). Maximum PTV coverage by 70 Gy was 33%. Maximum PTV coverage by 60 Gy was 77.5%. The following organ at risk (OAR) constraints were achieved for 90% of generated plans: Duodenum V20 < 30 cc, V30 < 3 cc, V35 < 1 cc; Small Bowel V20 < 15 cc, V30 < 1 cc, V35 < 0.1 cc; Stomach V20 < 20 cc, V30 < 2 cc, V35 < 1 cc. V40 < 0.5 cc was achieved for all OAR. Conclusions: Dose escalation to 60 Gy is dosimetrically feasible with adequate GTV coverage. The identified constraints for OAR's will be used in ongoing clinical trials.

Purpose: To review acute and late toxicities after chemoradiation for locally advanced pancreatic ductal adenocarcinoma in patients who were treated with escalated dose radiation (EDR). Methods and materials: Maximum Common Terminology Criteria for Adverse Events Version 4.0 acute toxicities (AT) during radiation and within 60 days after radiation were recorded for both acute gastrointestinal toxicity and overall toxicity (OT). Late toxicities were also recorded. EDR was generally delivered with daily image guidance and breath-hold techniques using intensity modulated radiation therapy (IMRT) planning. These were compared with patients who received standard dose radiation (SDR) delivered as 50.4 Gy in 28 fractions using 3-dimensional chemoradiation therapy planning. Results: A total of 59 of 154 patients (39%) received EDR with biologically equivalent doses >70 Gy. The most frequent schedules were 63 Gy in 28 fractions (19 of 154 patients), 67.5 Gy in 15 fractions (10 of 154 patients), and 70 Gy in 28 fractions (15 of 154 patients). No grade 4 or grade 5 OT or late toxicities were reported. Rates of grade 3 acute gastrointestinal toxicity were significantly lower in patients who received EDR compared with SDR (1% vs 14%; P < .001). Similarly, rates of grade 3 OT were also lower for EDR compared with SDR (4% vs 16%; P = .004). The proportion of patients who experienced no AT was higher in the EDR group than the SDR group (36% vs 15%; P = .001). For EDR patients treated with IMRT, a lower risk of AT was associated with a later treatment year (P = .007), nonpancreatic head tumor location (P = .01), breath-hold (P = .002), 4-dimensional computed tomography (P = .003), computed tomography on rails (P = .002), and lower stomach V40 (P = .03). With a median time of 12 months (range, 1-79 months) from the start of radiation therapy to the last known follow-up in the EDR group, 51 of 59 patients (86%) had no late toxicity. Six of 59 EDR patients (10%) had either strictures or gastrointestinal bleeding that required intervention. No significant predictors of late toxicity were identified. Conclusion: Overall acute and late toxicity rates were low with EDR using an IMRT technique with image guidance and respiratory gating.