Development of In Vitro Assays for Advancing Radioimmunotherapy against Brain Tumors

Abstract

Glioblastoma (GBM) is the most common primary brain tumor. Due to high resistance to treatment, local invasion, and a high risk of recurrence, GBM patient prognoses are often dismal, with median survival around 15 months. The current standard of care is threefold: surgery, radiation therapy, and chemotherapy with temozolomide (TMZ). However, patient survival has only marginally improved. Radioimmunotherapy (RIT) is a fourth modality under clinical trials and aims at combining immunotherapeutic agents with radiotherapy. Here, we develop in vitro assays for the rapid evaluation of RIT strategies. Using a standard cell irradiator and an Electric Cell Impedance Sensor, we quantify cell migration following the combination of radiotherapy and chemotherapy with TMZ and RIT with durvalumab, a PD-L1 immune checkpoint inhibitor. We measure cell survival using a cloud-based clonogenic assay. Irradiated T98G and U87 GBM cells migrate significantly (p < 0.05) more than untreated cells in the first 20–40 h post-treatment. Addition of TMZ increases migration rates for T98G at 20 Gy (p < 0.01). Neither TMZ nor durvalumab significantly change cell survival in 21 days post-treatment. Interestingly, durvalumab abolishes the enhanced migration effect, indicating possible potency against local invasion. These results provide parameters for the rapid supplementary evaluation of RIT against brain tumors.

Simulated Microgravity-Induced Changes to Drug Response in Cancer Cells Quantified Using Fluorescence Morphometry

“…The K562 cells grow in suspension. We followed specific protocols from ATCC in culturing both cell types, as reported in our previous works [ 12 , 40 , 42 , 43 ]. Briefly, we cultured K562 cells using RPMI 1640 (11875093, Life Technologies, Waltham, MA, USA), supplemented with 10% ( v / v ) fetal bovine serum (FBS) and 1% penicillin/streptomycin as growth medium.…”

Microfluidic Microcirculation Mimetic for Exploring Biophysical Mechanisms of Chemotherapy-Induced Metastasis

“…However, fluorescence-guided morphometry independent of the MMM was performed using a Zeiss Vert.A1 AXIO fluorescence microscope (Carl Zeiss, Jena, Germany) equipped with red, green and blue filters and with an inserted USB microscope camera (AmScope MU300, Ningbo, China), along with its AmScope (86x) image capture program. Details of the advanced fluorometric morphometry performed which yielded information on cytoplasmic cross section and nuclear cross section, following chemotherapeutic interventions, can be found in our recent publications [ 57 , 58 ]. Briefly, the fluorescent dyes Hoechst 33342 (ThermoFisher Scientific, Waltham, MA, USA), at a 0.1 mM final concentration, and Calcein AM (Invitrogen by ThermoFisher Scientific, Waltham, MA, USA), at a final concentration of 1 µg/mL, were used to stain the nucleus and the cytoplasm, respectively, for imaging.…”

Cellular Biophysical Markers for Rapid Evaluation of Immune Checkpoint Inhibitors in Radioimmunotherapy Against Glioblastomas

“…The immune checkpoint blockers, durvalumab (anti-PD-L1) 41 , and pembrolizumab (anti-PD-1) 42 in our in vitro assays, lead to several cellular and molecular level differences 38 in their direct action on GBM cell lines, T98G and U87, even in the absence of immune cells. Interestingly, radiation alone as well as pembrolizumab with radiation, both reduced (p < 0.05) cell-cell adhesion.…”

“…To address this question, we have developed and published in vitro assays which use biophysical parameters to quantify changes to cell behavior and cell death for both standard-of-care therapy, namely, temozolomide, (TMZ) against GBM, and radioimmunotherapy with durvalumab 48 , which is undergoing phase I/II clinical trials. Durvalumab, a PD-L1 inhibitor, did not alter cell migration as both radiation and TMZ did for the two glioblastoma cell lines (T98G and U87) tested.…”

“…In the present work, we have carried out similar molecular and cellular level investigations using pembrolizumab, a PD-1 inhibitor. Motivated by previous research indicating that certain chemotherapeutic agents and radiation may inadvertently promote metastasis [49][50][51][52] and having seen some of the cellular implications from research on the combination of immunotherapeutic agent, durvalumab 52 , and radiation, we conducted rigorous in-vitro assays to assess the cellular-level impact of pembrolizumab within the context of radioimmunotherapy for glioblastoma. Our study enabled us to simultaneously evaluate changes in various cellular characteristics, including cell death, motility, adhesion, and morphology, in real-time, following treatment.…”