In situ tumour arrays reveal early environmental control of cancer immunity.

The immune phenotype of a tumour is a key predictor of its response to immunotherapy1-4. Patients who respond to checkpoint blockade generally present with immune-inflamed5-7 tumours that are highly infiltrated by T cells. However, not all inflamed tumours respond to therapy, and even lower response rates occur among tumours that lack T cells (immune desert) or that spatially exclude T cells to the periphery of the tumour lesion (immune excluded)8. Despite the importance of these tumour immune phenotypes in patients, little is known about their development, heterogeneity or dynamics owing to the technical difficulty of tracking these features in situ. Here we introduce skin tumour array by microporation (STAMP)-a preclinical approach that combines high-throughput time-lapse imaging with next-generation sequencing of tumour arrays. Using STAMP, we followed the development of thousands of arrayed tumours in vivo to show that tumour immune phenotypes and outcomes vary between adjacent tumours and are controlled by local factors within the tumour microenvironment. Particularly, the recruitment of T cells by fibroblasts and monocytes into the tumour core was supportive of T cell cytotoxic activity and tumour rejection. Tumour immune phenotypes were dynamic over time and an early conversion to an immune-inflamed phenotype was predictive of spontaneous or therapy-induced tumour rejection. Thus, STAMP captures the dynamic relationships of the spatial, cellular and molecular components of tumour rejection and has the potential to translate therapeutic concepts into successful clinical strategies.

Smart lattice light-sheet microscopy for imaging rare and complex cellular events.

Light-sheet microscopes enable rapid high-resolution imaging of biological specimens; however, biological processes span spatiotemporal scales. Moreover, long-term phenotypes are often instigated by rare or fleeting biological events that are difficult to capture with a single imaging modality. Here, to overcome this limitation, we present smartLLSM, a microscope that incorporates artificial intelligence-based instrument control to autonomously switch between epifluorescent inverted imaging and lattice light-sheet microscopy (LLSM). We apply this approach to two unique processes: cell division and immune synapse formation. In each context, smartLLSM provides population-level statistics across thousands of cells and autonomously captures multicolor three-dimensional datasets or four-dimensional time-lapse movies of rare events at rates that dramatically exceed human capabilities. From this, we quantify the effects of Taxol dose on spindle structure and kinetochore dynamics in dividing cells and of antigen strength on cytotoxic T lymphocyte engagement and lytic granule polarization at the immune synapse. Overall, smartLLSM efficiently detects rare events within heterogeneous cell populations and records these processes with high spatiotemporal four-dimensional imaging over statistically significant replicates.

An open-access volume electron microscopy atlas of whole cells and tissues.

Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometre resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations in that they visualize only a single slice or a relatively small volume of the cell, respectively. Focused ion beam-scanning electron microscopy (FIB-SEM) has demonstrated the ability to image small volumes of cellular samples with 4-nm isotropic voxels1. Owing to advances in the precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning, we have increased the volume that can be imaged with 4-nm voxels by two orders of magnitude. Here we present a volume EM atlas at such resolution comprising ten three-dimensional datasets for whole cells and tissues, including cancer cells, immune cells, mouse pancreatic islets and Drosophila neural tissues. These open access data (via OpenOrganelle2) represent the foundation of a field of high-resolution whole-cell volume EM and subsequent analyses, and we invite researchers to explore this atlas and pose questions.

Actin depletion initiates events leading to granule secretion at the immunological synapse.

Cytotoxic T lymphocytes (CTLs) use polarized secretion to rapidly destroy virally infected and tumor cells. To understand the temporal relationships between key events leading to secretion, we used high-resolution 4D imaging. CTLs approached targets with actin-rich projections at the leading edge, creating an initially actin-enriched contact with rearward-flowing actin. Within 1 min, cortical actin reduced across the synapse, T cell receptors (TCRs) clustered centrally to form the central supramolecular activation cluster (cSMAC), and centrosome polarization began. Granules clustered around the moving centrosome within 2.5 min and reached the synapse after 6 min. TCR-bearing intracellular vesicles were delivered to the cSMAC as the centrosome docked. We found that the centrosome and granules were delivered to an area of membrane with reduced cortical actin density and phospholipid PIP2. These data resolve the temporal order of events during synapse maturation in 4D and reveal a critical role for actin depletion in regulating secretion.

Cardiotoxicity of Anti-Cancer Radiation Therapy: a Focus on Heart Failure.

PURPOSE OF REVIEW: As the percentage of patients achieving long-term survival following treatment of their cancer grows, it is increasingly important to understand the long-term toxicities of cancer-directed treatment. In this review, we highlight the recent findings regarding radiation-induced cardiotoxicity across multiple disease sites, with a particular focus on heart failure. RECENT FINDINGS: Despite its relative lack of study historically, radiation-induced heart failure has now recently been implicated in several studies of breast cancer, lung cancer, esophageal cancer, and lymphoma as a non-trivial potential consequence of thoracic radiotherapy. Data regarding specific cardiac dosimetric endpoints relevant to cardiotoxicity continue to accumulate. Radiation-induced heart failure is a rare but significant toxicity of thoracic radiotherapy, that is likely underreported. Important areas for future focus include understanding the interplay between thoracic radiotherapy and concurrent cardiotoxic systemic therapy as well as development of potential mitigation strategies and novel therapeutics.

Comfort Level of US Radiation Oncology Graduates: Assessment of Transition to Independent Clinical Practice.

Radiation training programs are designed to prepare graduates for independent practice, with metrics in place to assess appropriateness of clinical decision-making. Here, we investigated the self-assessed preparedness of US graduates during the transition to independent practice.An anonymous, Internet-based survey was distributed to recent graduates of radiation oncology residencies (2016-2017). A Likert scale was used to assess comfort with various aspects of practice, as well as "time" to development of comfort in independent practice.Responses were obtained from 70/210 (33%), the majority reported training in programs with 5-8 residents (n = 35). Most (77%) reported designing between 500 and 900 treatment plans during training (n = 54). Only 41% of respondents reported the opportunity to review treatment plans and make decisions about safety/adequacy without attending input > 50% of the time (n = 29). Thirty percent of residents reported being responsible for seeing/managing on-treatment visits (OTVs) ≤ 75% of the time. Aspects with which practitioners reported the least comfort were understanding of billing/application to practice (2.43, IQR 2-3), orthovoltage (superficial radiation) setup and field design (2.57, IQR 1-4), and planning/delivery of prostate implants (2.82, IQR 2-4). Increased mean comfort levels were reported by those designing > 700 treatment plans in training as well as those reporting an opportunity to evaluate plans and make clinical decisions prior to attending input > 50% of the time during residency. Comfort with the delivery of stereotactic body radiation (SBRT) correlated with caseload for liver, spine, prostate, and CNS disease sites but not lung.Variations in training experiences exist across institutions. Here, a lower than expected number of residents reported seeing/managing OTVs as well as reviewing treatment plans prior to attending input during training. Overall comfort was correlated with case volume and opportunities to independently review treatment plans prior to attending input. These data highlight areas of opportunity for improving resident education with implications for ease of transition to independent clinical practice.

Cortical actin recovery at the immunological synapse leads to termination of lytic granule secretion in cytotoxic T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) eliminate virally infected cells through directed secretion of specialized lytic granules. Because a single CTL can kill multiple targets, degranulation must be tightly regulated. However, how CTLs regulate the termination of granule secretion remains unclear. Previous work demonstrated that centralized actin reduction at the immune synapse precedes degranulation. Using a combination of live confocal, total internal reflection fluorescence, and superresolution microscopy, we now show that, after granule fusion, actin recovers at the synapse and no further secretion is observed. Depolymerization of actin led to resumed granule secretion, suggesting that recovered actin acts as a barrier preventing sustained degranulation. Furthermore, RAB27a-deficient CTLs, which do not secrete cytotoxic granules, failed to recover actin at the synapse, suggesting that RAB27a-mediated granule secretion is required for actin recovery. Finally, we show that both actin clearance and recovery correlated with synaptic phosphatidylinositol 4,5-bisphosphate (PIP2) and that alterations in PIP2 at the immunological synapse regulate cortical actin in CTLs, providing a potential mechanism through which CTLs control cortical actin density. Our work provides insight into actin-related mechanisms regulating CTL secretion that may facilitate serial killing during immune responses.

The role of the cytoskeleton at the immunological synapse.

It has been over 30 years since the reorganization of both the microtubule network and a 'peculiar actin polarization' was reported at the contact area of cytotoxic T lymphocytes interacting with target cells. Since that time, hundreds of studies have been published in an effort to elucidate the structure and function of the microtubule network and the actin cytoskeleton in T-cell activation, migration, and effector function at the interface between a T cell and its cognate antigen-presenting cell or target cell. This interface has become known as the immunological synapse, and this review examines some of the roles played by the cytoskeleton at the synapse.

A systematic comparison of mathematical models for inherent measurement of ciliary length: how a cell can measure length and volume.

Cells control organelle size with great precision and accuracy to maintain optimal physiology, but the mechanisms by which they do so are largely unknown. Cilia and flagella are simple organelles in which a single measurement, length, can represent size. Maintenance of flagellar length requires an active transport process known as intraflagellar transport, and previous measurements suggest that a length-dependent feedback regulates intraflagellar transport. But the question remains: how is a length-dependent signal produced to regulate intraflagellar transport appropriately? Several conceptual models have been suggested, but testing these models quantitatively requires that they be cast in mathematical form. Here, we derive a set of mathematical models that represent the main broad classes of hypothetical size-control mechanisms currently under consideration. We use these models to predict the relation between length and intraflagellar transport, and then compare the predicted relations for each model with experimental data. We find that three models-an initial bolus formation model, an ion current model, and a diffusion-based model-show particularly good agreement with available experimental data. The initial bolus and ion current models give mathematically equivalent predictions for length control, but fluorescence recovery after photobleaching experiments rule out the initial bolus model, suggesting that either the ion current model or a diffusion-based model is more likely correct. The general biophysical principles of the ion current and diffusion-based models presented here to measure cilia and flagellar length can be generalized to measure any membrane-bound organelle volume, such as the nucleus and endoplasmic reticulum.

Hypofractionated Radiation Therapy: A Cross-sectional Survey Study of US Radiation Oncologists.

OBJECTIVES: For many malignancies, hypofractionated radiotherapy (HFRT) is an accepted standard associated with decreased treatment time and costs. United States provider beliefs regarding HFRT likely impact its adoption but are poorly studied. We surveyed US-based radiation oncologists (ROs) to gauge HFRT utilization rates for prostate (PC), breast (BC), and rectal cancer (RC) and to characterize the beliefs governing these decisions. METHODS: From July to October 2021, an anonymized, online survey was electronically distributed to ROs actively practicing in the United States. Demographic and practice characteristic information was collected. Questions assessing rates of offering HFRT for PC, BC, and RC and perceived limitations towards using HFRT were administered. RESULTS: A total of 203 eligible respondents (72% male, 72% White, 53% nonacademic practice, 69% with 11+ years in practice) were identified. Approximately 50% offered stereotactic body radiation therapy (SBRT) for early/favorable intermediate risk PC. Although >90% of ROs offered whole-breast HFRT for early-stage BC, only 33% offered accelerated partial-breast irradiation (APBI). Overall, 41% of ROs offered short-course neoadjuvant RT for RC. The primary reported barriers to HFRT utilization were lack of data, inexperience, and referring provider concerns. CONCLUSIONS: HFRT is safe, effective, and beneficial, yet underutilized-particularly prostate SBRT, APBI, and short-course RT for RC. Skills retraining and education of ROs and referring providers may increase utilization rates.

Advancing Beyond the Hippocampus to Preserve Cognition for Patients With Brain Metastases: Dosimetric Results From a Phase 2 Trial of Memory-Avoidance Whole Brain Radiation Therapy.

Purpose: Recent advances to preserve neurocognitive function in patients treated for brain metastases include stereotactic radiosurgery, hippocampal avoidance whole brain radiation therapy (WBRT), and memantine administration. The hippocampus, corpus callosum, fornix, and amygdala are key neurocognitive substructures with a low propensity for brain metastases. Herein, we report our preliminary experience using a "memory-avoidance" WBRT (MA-WBRT) approach that spares these substructures for patients with >15 brain metastases. Methods and Materials: Ten consecutive patients treated with MA-WBRT on a phase 2 clinical trial were reviewed. In each patient, the hippocampi, amygdalae, corpus callosum, and fornix were contoured. Patients were not eligible for MA-WBRT if they had metastases in these substructures. A memory-avoidance region was created using a 5-mm volumetric expansion around these substructures. Hotspots were avoided in the hypothalamus and pituitary gland. Coverage of brain metastases was prioritized over memory avoidance dose constraints. Dose constraints for these avoidance structures included a D100% ≤ 9 Gy and D0.03 cm3 ≤ 16 Gy (variation acceptable to 20 Gy). LINAC-based volumetric modulated arc therapy plans were generated for a prescription dose of 30 Gy in 10 fractions. Results: On average, the memory avoidance structure volume was 37.1 cm3 (range, 25.2-44.6 cm3), occupying 2.5% of the entire whole brain target volume. All treatment plans met the D100% dose constraint, and 8 of 10 plans met the D0.03 cm3 constraint, with priority given to tumor coverage for the remaining 2 cases. Target coverage (D98% > 25 Gy) and homogeneity (D2% ≤ 37.5 Gy) were achieved for all plans. Conclusions: Modern volumetric modulated arc therapy techniques allow for sparing of the hippocampus, amygdala, corpus callosum, and fornix with good target coverage and homogeneity. After enrollment is completed, quality of life and cognitive data will be evaluated to assess the efficacy of MA-WBRT to mitigate declines in quality of life and cognition after whole brain radiation.

A role for Rab7 in the movement of secretory granules in cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) are potent killers of virally infected and tumorigenic cells. Upon recognition of target cells, CTL undergo polarized secretion of secretory lysosomes at the immunological synapse (IS) that forms between CTL and target. However, the molecular machinery involved in the polarization of secretory lysosomes is still largely uncharacterized. In this paper, we investigated the role of Rab7 in the polarization of secretory lysosomes. We show that silencing of Rab7 by RNA interference reduces the ability of CTL to kill targets. GTP-bound Rab7 and Rab interacting lysosomal protein, RILP, interact and both localize to secretory lysosomes in CTL. Over-expression of RILP recruits dynein to the membranes of secretory lysosomes and triggers their movement toward the centrosome. Together, these results suggest that Rab7 may play a role in secretory lysosome movement toward the centrosome by interacting with RILP to recruit the minus-end motor, dynein.

Cytotoxic T lymphocyte effector function is independent of nucleus-centrosome dissociation.

Cytotoxic T lymphocytes (CTLs) kill tumorigenic and virally infected cells by targeted secretion of lytic granule contents. The precise point at which secretion occurs is directed by the centrosome docking at the immunological synapse (IS). The centrosome is highly dynamic in CTLs, lagging behind the nucleus in the uropod of migrating CTLs, but translocating across the entire length of the cell to dock at the IS when a target cell is recognized. While in most cell types, the centrosome is always closely associated with the nuclear membrane, in CTLs, it often appears to be dissociated from the nucleus, both in migrating cells and when forming an IS. We asked whether this dissociation is required for CTL killing, by expressing GFP-BICD2-NT-nesprin-3, which tethers the centrosome to the nucleus irreversibly. Immunofluorescence microscopy revealed that the centrosome polarized successfully to the central supramolecular activation complex (cSMAC) of the synapse in GFP-BICD2-NT-nesprin-3-expressing CTLs, with the centrosome and nucleus migrating together to the IS. CTLs in which the centrosome was "glued" to the nucleus were able to dock and release granules at the IS as effectively as mock-treated cells. These data demonstrate that CTL cytotoxicity is independent of centrosomal dissociation from the nuclear envelope.

Intraoperative Radiation Therapy for Gastrointestinal Malignancies.

Despite improvements in definitive therapy, many patients with gastrointestinal malignancies experience local recurrences or have unresectable disease making subsequent management often challenging and morbid. Although higher doses of radiation may offer improved local control, the ability for dose escalation of external beam radiation therapy is often limited by adjacent radiosensitive structures. Intraoperative radiation therapy allows for additional radiotherapy to be delivered directly to the tumor or areas at highest risk for local recurrence while minimizing toxicity to adjacent structures, offering potentially improved outcomes for patients with unresectable disease or those with a high risk of local recurrence.

Therapeutic Cancer Vaccines for the Management of Recurrent and Metastatic Head and Neck Cancer: A Review.

Importance: Squamous cell carcinoma of the head and neck (HNSCC) is prevalent globally and in the US. Management, particularly after disease recurrence, can be challenging, and exploring additional treatment modalities, such as therapeutic cancer vaccines, may offer an opportunity to improve outcomes in this setting. Observations: This review provides an overview of the clinical efficacy of different treatment modalities that are currently available for the treatment of recurrent and metastatic HNSCC, including checkpoint inhibitors and targeted therapies, with a detailed summary of the numerous T-cell vaccines that have been studied in the setting of HNSCC, as well as a detailed summary of B-cell therapeutic vaccines being investigated for various malignant tumors. Conclusions and Relevance: The findings of this review suggest that several therapeutic T-cell and B-cell vaccines, which have been recently developed and evaluated in a clinical setting, offer a promising treatment modality with the potential to improve outcomes for patients with recurrent and metastatic HNSCC.

Prostate brachytherapy utilization in the COVID-19 era: A cross-sectional study of radiation oncologists in the United States.

PURPOSE: Despite advantages such as abbreviated treatment course, brachytherapy (BT) utilization rates for prostate cancer (PC) in the United States (US) are declining. We surveyed practicing US radiation oncologists (ROs) to determine the proportion who offer BT for PC and whether the COVID-19 pandemic influenced practice patterns. MATERIALS AND METHODS: From July-October 2021, we surveyed practicing US ROs. Provider demographic and practice characteristics were collected. Questions assessing utilization of BT and external beam (EBRT) for patients of varying risk groups and the effect of the pandemic on practice patterns were administered. Descriptive statistics were reported. The bivariate relationships between provider characteristics and likelihood of offering BT were assessed using the Chi-square test (α < 0.05). RESULTS: Six percent of surveyed ROs responded, with 203 meeting inclusion criteria (72% male, 72% white, 53% non-academic, 69% >10 years in practice) and 156 (77%) treating PC. For low-risk, fewer providers offered BT (41% total; 25% low dose rate [LDR], 10% high dose rate [HDR], 6% both) than stereotactic body (SBRT) (54%) and moderately hypofractionated radiation therapy (MHFRT) (83%). For favorable intermediate risk, fewer offered BT (37% total; 21% LDR, 10% HDR, 6% both) than SBRT (48%), MHFRT (87%), and conventionally fractionated EBRT (38%). For high (44%) and very-high (37%) risk, fewer offered EBRT+BT than EBRT alone. For every risk group, academic ROs were significantly more likely to offer BT (all p-values<0.05). <1% of respondents reported increased pandemic-related BT usage. CONCLUSIONS: US ROs, particularly in non-academic settings, do not routinely offer BT monotherapy or boost (<50%). Practice patterns were unaffected by COVID-19. Retraining may be critical to increasing utilization.

Factors Associated with Total Laryngectomy Utilization in Patients with cT4a Laryngeal Cancer.

BACKGROUND: Despite recommendations for upfront total laryngectomy (TL), many patients with cT4a laryngeal cancer (LC) instead undergo definitive chemoradiation, which is associated with inferior survival. Sociodemographic and oncologic characteristics associated with TL utilization in this population are understudied. METHODS: This retrospective cohort study utilized hospital registry data from the National Cancer Database to analyze patients diagnosed with cT4a LC from 2004 to 2017. Patients were stratified by receipt of TL, and patient and facility characteristics were compared between the two groups. Logistic regression analyses and Cox proportional hazards methodology were performed to determine variables associated with receipt of TL and with overall survival (OS), respectively. OS was estimated using the Kaplan-Meier method and compared between treatment groups using log-rank testing. TL usage over time was assessed. RESULTS: There were 11,149 patients identified. TL utilization increased from 36% in 2004 to 55% in 2017. Treatment at an academic/research program (OR 3.06) or integrated network cancer program (OR 1.50), male sex (OR 1.19), and Medicaid insurance (OR 1.31) were associated with increased likelihood of undergoing TL on multivariate analysis (MVA), whereas age > 61 (OR 0.81), Charlson-Deyo comorbidity score ≥ 3 (OR 0.74), and clinically positive regional nodes (OR 0.78 [cN1], OR 0.67 [cN2], OR 0.21 [cN3]) were associated with decreased likelihood. Those undergoing TL with post-operative radiotherapy (+/- chemotherapy) had better survival than those receiving chemoradiation (median OS 121 vs. 97 months; p = 0.003), and TL + PORT was associated with lower risk of death compared to chemoradiation on MVA (HR 0.72; p = 0.024). CONCLUSIONS: Usage of TL for cT4a LC is increasing over time but remains below 60%. Patients seeking care at academic/research centers are significantly more likely to undergo TL, highlighting the importance of decreasing barriers to accessing these centers. Increased focus should be placed on understanding and addressing the additional patient-, physician-, and system-level factors that lead to decreased utilization of surgery.

Novel Intrafraction Motion Tracking During Postoperative Spine Stereotactic Irradiation for a Patient With Carbon Fiber Fixation Hardware.

Carbon-fiber reinforced (CFR) polyetheretherketone hardware is an alternative to traditional metal hardware used for spinal fixation surgeries before postoperative radiation therapy for patients with spinal metastases. CFR hardware's radiolucency decreases metal artifact, improving visualization and accuracy of treatment planning. We present the first clinical use and proof of principle of CFR spinal hardware with tantalum markers used for successful tracking of intrafraction motion (IM) using Varian TrueBeam IMR (Intrafraction Motion Review) software module during postoperative spine stereotactic radiation. A 63-year-old woman with history of endometrial cancer presented with acute back pain. Imaging demonstrated pathologic T12 vertebral fracture with cord compression. She underwent T12 vertebrectomy with circumferential decompression and posterior instrumented T10-L2 fusion at our facility using CFR-polyetheretherketone hardware with tantalum screw markers followed by postoperative stereotactic body radiation therapy to 3000 cGy in 5 fractions delivered to T11-T12. Tantalum screw markers were used for IMR tracking. During irradiation, 260 kV images were acquired, and IMR software was able to identify and track markers. During the entire treatment, the IM motions were less than 3 mm. This is the first presented case of CFR spinal hardware with tantalum markers used for successful IMR tracking of IM during daily spine stereotactic treatment. Future work will be needed to improve workflow and create a spine-specific IMR protocol.

ESCRT-mediated membrane repair protects tumor-derived cells against T cell attack.

Cytotoxic T lymphocytes (CTLs) and natural killer cells kill virus-infected and tumor cells through the polarized release of perforin and granzymes. Perforin is a pore-forming toxin that creates a lesion in the plasma membrane of the target cell through which granzymes enter the cytosol and initiate apoptosis. Endosomal sorting complexes required for transport (ESCRT) proteins are involved in the repair of small membrane wounds. We found that ESCRT proteins were precisely recruited in target cells to sites of CTL engagement immediately after perforin release. Inhibition of ESCRT machinery in cancer-derived cells enhanced their susceptibility to CTL-mediated killing. Thus, repair of perforin pores by ESCRT machinery limits granzyme entry into the cytosol, potentially enabling target cells to resist cytolytic attack.