PurposeAlthough vascular alterations in solid tumor malignancies are known to decrease therapeutic delivery, the effects of leukemia-induced bone marrow vasculature (BMV) alterations on therapeutic delivery are not well known. Additionally, functional quantitative measurements of the leukemic BMV during chemotherapy and radiation therapy are limited, largely due to a lack of high-resolution imaging techniques available preclinically. This study develops a murine model using compartmental modeling for quantitative multiphoton microscopy (QMPM) to characterize the malignant BMV before and during treatment.Methods and MaterialsUsing QMPM, live time-lapsed images of dextran leakage from the local BMV to the surrounding bone marrow of mice bearing acute lymphoblastic leukemia (ALL) were taken and fit to a 2-compartment model to measure the transfer rate (Ktrans), fractional extracellular extravascular space (νec), and vascular permeability parameters, as well as functional single-vessel characteristics. In response to leukemia-induced BMV alterations, the effects of 2 to 4 Gy low-dose radiation therapy (LDRT) on the BMV, drug delivery, and mouse survival were assessed post-treatment to determine whether neoadjuvant LDRT before chemotherapy improves treatment outcome.ResultsMice bearing ALL had significantly altered Ktrans, increased νec, and increased permeability compared with healthy mice. Angiogenesis, decreased single-vessel perfusion, and decreased vessel diameter were observed. BMV alterations resulted in disease-dependent reductions in cellular uptake of Hoechst dye. LDRT to mice bearing ALL dilated BMV, increased single-vessel perfusion, and increased daunorubicin uptake by ALL cells. Consequently, LDRT administered to mice before receiving nilotinib significantly increased survival compared with mice receiving LDRT after nilotinib, demonstrating the importance of LDRT conditioning before therapeutic administration.ConclusionThe developed QMPM enables single-platform assessments of the pharmacokinetics of fluorescent agents and characterization of the BMV. Initial results suggest BMV alterations after neoadjuvant LDRT may contribute to enhanced drug delivery and increased treatment efficacy for ALL. The developed QMPM enables observations of the BMV for use in ALL treatment optimization. Although vascular alterations in solid tumor malignancies are known to decrease therapeutic delivery, the effects of leukemia-induced bone marrow vasculature (BMV) alterations on therapeutic delivery are not well known. Additionally, functional quantitative measurements of the leukemic BMV during chemotherapy and radiation therapy are limited, largely due to a lack of high-resolution imaging techniques available preclinically. This study develops a murine model using compartmental modeling for quantitative multiphoton microscopy (QMPM) to characterize the malignant BMV before and during treatment. Using QMPM, live time-lapsed images of dextran leakage from the local BMV to the surrounding bone marrow of mice bearing acute lymphoblastic leukemia (ALL) were taken and fit to a 2-compartment model to measure the transfer rate (Ktrans), fractional extracellular extravascular space (νec), and vascular permeability parameters, as well as functional single-vessel characteristics. In response to leukemia-induced BMV alterations, the effects of 2 to 4 Gy low-dose radiation therapy (LDRT) on the BMV, drug delivery, and mouse survival were assessed post-treatment to determine whether neoadjuvant LDRT before chemotherapy improves treatment outcome. Mice bearing ALL had significantly altered Ktrans, increased νec, and increased permeability compared with healthy mice. Angiogenesis, decreased single-vessel perfusion, and decreased vessel diameter were observed. BMV alterations resulted in disease-dependent reductions in cellular uptake of Hoechst dye. LDRT to mice bearing ALL dilated BMV, increased single-vessel perfusion, and increased daunorubicin uptake by ALL cells. Consequently, LDRT administered to mice before receiving nilotinib significantly increased survival compared with mice receiving LDRT after nilotinib, demonstrating the importance of LDRT conditioning before therapeutic administration. The developed QMPM enables single-platform assessments of the pharmacokinetics of fluorescent agents and characterization of the BMV. Initial results suggest BMV alterations after neoadjuvant LDRT may contribute to enhanced drug delivery and increased treatment efficacy for ALL. The developed QMPM enables observations of the BMV for use in ALL treatment optimization.
Malignant gliomas (MG) are rapidly fatal despite multimodal treatments including radiation therapy, used to treat nearly all MG patients, or even the emerging cellular immunotherapies. Therapeutic resistance in glioma is at least partly related to tolerogenic STAT3 activity in both glioma cancer stem cells (GCSs) and in the tumor-associated myeloid immune cells, such as macrophages and microglia, which dominate MG microenvironment. We previously demonstrated that STAT3 activity in GSCs and tumor-associated myeloid cells can be targeted using Toll-like Receptor-9 (TLR9)-targeted oligonucleotide therapeutics such as siRNA or antisense oligonucleotides (ASO).1–3
Methods
Here, we describe development of a new TLR9-targeted and double-stranded STAT3 antisense oligonucleotide (CpG-STAT3dsASO) with optimized efficacy and tolerability for glioma immunotherapy.
Results
Compared to our benchmark ASO oligonucleotides, the locked nucleic acid (LNA)-modified CpG-STAT3dsASO showed enhanced STAT3 knockdown in human and in mouse glioma cells and also in TLR9+ immune cells, such as macrophages and microglia. When tested against orthotopic model of human U251 glioma, intracranial injections of CpG-STAT3dsASO (1 mg/kg/q2w) inhibited tumor growth and significantly extended survival of immunodeficient NSG mice compared to benchmark oligonucleotide. Next, we tested the efficacy of CpG-STAT3dsASO against syngeneic GL261 or QPP8 glioma in immunocompetent mice.4 Our initial results demonstrated that CpG-STAT3dsASO was more effective and significantly better tolerated than single-stranded CpG-STAT3ASO when injected intracranially. All tested CpG-STAT3ASO variants induced activation of intratumoral DCs, macrophages and microglia, while reducing numbers of tumor-associated macrophages (TAMs), resting microglia and regulatory T cells as assessed using flow cytometry. However, the intratumoral recruitment of effector CD8 T cells was limited. To improve CD8 T cell activation, we next combined intracranial CpG-STAT3dsASO administration at low 0.25 mg/kg dosing with systemic PD1 blockade. The anti-PD1/CpG-STAT3dsASO combination triggered complete rejection of both orthotopic GL261 and the PD1-refractory QPP8 tumors in the majority of treated mice, while neither treatment was curative alone. Importantly, Our single-cell transcriptomic analysis and spatial profiling of the brain sections from all treatment groups confirmed the complementary effect of CpG-STAT3dsASO and PD1 blockade on the glioma microenvironment. CpG-STAT3dsASO reprogrammed glioma-associated myeloid cells into antigen-presenting cells and phagocytes, expanded Th1 CD4 lymphocytes while reducing Treg percentages. These conditions unlocked the potential of PD1 blockade to recruit effector CD8 T cells into glioma without indication of lymphocyte exhaustion.
Conclusions
Our results underscore the potential of using myeloid cell-targeted CpG-STAT3dsASO to overcome glioma immune evasion and thereby to sensitize tumors to PD1 immune checkpoint blockade and potentially other T-cell based cancer immunotherapies.
Acknowledgements
This work was supported by the NCI/NIH award R01CA215183 (to M.K.). The content is the responsibility of the authors and does not necessarily represent the official views of the NIH.
References
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This work reports our initial experience using multimodality image guidance to improve total marrow irradiation (TMI) using helical tomotherapy. We also monitored the details of the treatment delivery to glean information necessary for the implementation of future adaptive processes. A patient with metastatic Ewing's sarcoma underwent MRI, and bone scan imaging prior to TMI. A whole body kilovoltage CT (kVCT) scan was obtained for intensity modulated TMI treatment planning, including a boost treatment to areas of bony involvement. The delivered dose was estimated by using MVCT images from the helical tomotherapy treatment unit, compared to the expected dose distributions mapped onto the kVCT images. Clinical concerns regarding patient treatment and dosimetric uncertainties were also evaluated. A small fraction of thoracic bone volume received lower radiation dose than the prescribed dose. Reconstructed planned treatment volume (PTV) and the dose delivered to the lung were identical to planned dose. Bone scan imaging had a higher sensitivity for detecting skeletal metastasis compared to MR imaging. However the bone scan lacked sufficient specificity in three dimensions to be useful for planning conformal radiation boost treatments. Inclusion of appropriate imaging modalities improves detection of metastases, which allows the possibility of a radiation dose boost to metastases during TMI. Conformal intensity modulated radiation therapy via helical tomotherapy permitted radiation delivery to metastases in the skull with reduced dose to brain in conjunction with TMI. While TMI reduces irradiation to the lungs, onboard megavoltage computed tomography (MVCT) to verify accurate volumetric dose coverage to marrow-containing thoracic bones may be essential for successful conformal TMI treatment.
Posttransplant cyclophosphamide (PTCy) platform has shown low rates of graft-versus-host disease (GVHD) and nonrelapse mortality (NRM) after haploidentical hematopoietic cell transplantation (HaploHCT). However, because of the limited disease control, relapse rate remains a major cause of treatment failure in high-risk patients. Total marrow and lymphoid irradiation (TMLI) allows for delivery of high radiation to bone marrow and other targeted structures, without increasing off-target radiation exposure and toxicity to end organs. In this phase 1 trial, 31 patients with high-risk and/or active primary refractory leukemias or myelodysplastic syndrome underwent peripheral blood stem cell HaploHCT with TMLI, fludarabine, and cyclophosphamide as the conditioning regimen. Radiation dose was escalated in increments of 200 cGy (1200-2000 cGy). GVHD prophylaxis was PTCy with tacrolimus/mycophenolate mofetil. Grade 2 toxicities by the Bearman scale were mucositis (n = 1), hepatic (n = 3), gastrointestinal (n = 5), and cardiac (n = 2). One patient (1800 cGy) experienced grade 3 pulmonary toxicity (dose-limiting toxicity). At a follow-up duration of 23.9 months for the whole cohort; 2-year NRM was 13%. Cumulative incidence of day 100 grade 2 to 4 and 3 to 4 acute GVHD was 52% and 6%, respectively. Chronic GVHD at 2 years was 35%. For patients treated with 2000 cGy, with a median follow-up duration of 12.3 months, 1-year relapse/progression, progression-free survival, and overall survival rates were 17%, 74%, and 83%, respectively. In conclusion, HaploHCT-TMLI with PTCy was safe and feasible in our high-risk patient population with promising outcomes.
Purpose TMI utilizes IMRT to deliver organ sparing targeted radiotherapy in patients undergoing hematopoietic cell transplantation (HCT). TMI addresses an unmet need, specifically patients with refractory or relapsed (R/R) hematologic malignancies who have poor outcomes with standard HCT regimens and where attempts to improve outcomes by adding or dose escalating TBI are not possible due to increased toxicities. Over 500 patients have received TMI at this center. This review summarizes this experience including planning and delivery, clinical results, and future directions. Methods Patients were treated on prospective allogeneic HCT trials using helical tomographic or VMAT IMRT delivery. Target structures included the bone/marrow only (TMI), or the addition of lymph nodes, and spleen (total marrow and lymphoid irradiation, TMLI). Total dose ranged from 12 to 20 Gy at 1.5-2.0 Gy fractions twice daily. Results Trials demonstrate engraftment in all patients and a low incidence of radiation related toxicities and extramedullary relapses. In R/R acute leukemia TMLI 20 Gy, etoposide, and cyclophosphamide (Cy) results in a 1-year non-relapse mortality (NRM) rate of 6% and 2-year overall survival (OS) of 48%; TMLI 12 Gy added to fludarabine (flu) and melphalan (mel) in older patients (≥ 60 years old) results in a NRM rate of 33% comparable to flu/mel alone, and 5-year OS of 42%; and TMLI 20 Gy/flu/Cy and post-transplant Cy (PTCy) in haplo-identical HCT results in a 2-year NRM rate of 13% and 1-year OS of 83%. In AML in complete remission, TMLI 20 Gy and PTCy results in 2-year NRM, OS, and GVHD free/relapse-free survival (GRFS) rates of 0%, 86·7%, and 59.3%, respectively. Conclusion TMI/TMLI shows significant promise, low NRM rates, the ability to offer myeloablative radiation containing regimens to older patients, the ability to dose escalate, and response and survival rates that compare favorably to published results. Collaboration between radiation oncology and hematology is key to successful implementation. TMI/TMLI represents a paradigm shift from TBI towards novel strategies to integrate a safer and more effective target-specific radiation therapy into HCT conditioning beyond what is possible with TBI and will help expand and redefine the role of radiotherapy in HCT.
We review the state-of-the-art in bone and marrow tissue engineering (BMTE) and hematological cancer tissue engineering (HCTE) in light of the recent interest in bone marrow environment and pathophysiology of hematological cancers. This review focuses on engineered BM tissue and organoids as in vitro models of hematological cancer therapeutics, along with identification of BM components and their integration as synthetically engineered BM mimetic scaffolds. In addition, the review details interaction dynamics of various BM and hematologic cancer (HC) cell types in co-culture systems of engineered BM tissues/phantoms as well as their relation to drug resistance and cytotoxicity. Interaction between hematological cancer cells and their niche, and the difference with respect to the healthy niche microenvironment narrated. Future perspectives of BMTE for in vitro disease models, BM regeneration and large scale ex vivo expansion of hematopoietic and mesenchymal stem cells for transplantation and therapy are explained. We conclude by overviewing the clinical application of biomaterials in BM and HC pathophysiology and its challenges and opportunities.
Hypofractionated stereotactic body radiotherapy treatments (SBRT) have improved patient treatment outcomes. Extensive studies were performed investigating how vascular changes during treatment affect its efficacy. Unfortunately, histology is unable to perform non-invasive longitudinal assessments by directly measuring blood perfusion. Here, we present a novel preclinical theranostic μCT-guided irradiator/Fluorescence Molecular Imager, designed to perform a fast, non-invasive, and longitudinal assessment of tumor vascular response (TVR) to targeted radiotherapy. Our technique allows rapid assessment of spatiotemporal differences in indocyanine green (ICG) kinetics in tumors using principal component (PC) analysis, before and after tumor irradiation. Results show that changes were observed in the normalized first and second PC feature pixel values (p=0.0559, 0.0432 paired t-test). Moreover, we implemented a fast PC-based classification algorithm that yields spatially-resolved TVR maps. Our first-of-its-kind theranostic system, allowing automated assessment of TVR to SBRT, will be used to better understand the role of tumor perfusion in metastasis and tumor control.
We developed a mathematical model to simulate the growth of tumor volume and its response to a single fraction of high dose irradiation. We made several key assumptions of the model. Tumor volume is composed of proliferating (or dividing) cancer cells and non-dividing (or dead) cells. Tumor growth rate (or tumor volume doubling time, Td) is proportional to the ratio of the volumes of tumor vasculature and the tumor. The vascular volume grows slower than the tumor by introducing the vascular growth retardation factor, theta. Upon irradiation the proliferating cells gradually die over a fixed time period after irradiation. Dead cells are cleared away with cell clearance time, Tcl. The model was applied to simulate pre-treatment growth and post-treatment radiation response of rat rhabdomyosarcoma tumor and metastatic brain tumors of five patients who were treated by Gamma Knife stereotactic radiosurgery (GKSRS). By selecting appropriate model parameters, we showed the temporal variation of the tumors for both the rat experiment and the clinical GKSRS cases could be easily replicated by the simple model. Additionally, the application of our model to the GKSRS cases showed that the alpha-value, which is an indicator of radiation sensitivity in the LQ model, and the retardation factor theta could be predictors of the post-treatment volume change. Since there is a large statistical uncertainty of this result due to the small sample size, a future clinical study with a larger number of patients is needed to confirm this finding.
