Anti-inflammatory combinatorial therapy to enhance killing efficacy with patient-derived preclinical models

Many chemotherapeutic drugs induce oxidative stress by accelerating the accumulation of reactive oxygen species (ROS), which triggers the death of cancer cells and then causes severe DNA damage in cancer cells. Here, we proposed using a preclinical microfluidic model to evaluate the combination of doxorubicin and aspirin (DA) for anti-inflammatory therapy using patient-derived circulating tumor cell (CTC) clusters. The preclinical model could perform high-throughput screening of drug combinations and used valves to regulate media inflow for CTC cluster formation. We demonstrated that low-dose aspirin (445–500 mg/ml) and a suboptimal dose of doxorubicin (0.5 D) for seven days could produce higher killing efficacy and significantly reduced the proportion of cancer stem cells and colony-forming ability. Compared with the treatment with doxorubicin alone, the intracellular oxidative activity in the sample under combinatorial DA treatment was reduced, as demonstrated by the intensity of Calcein AM. We demonstrated that the treatment outcomes were mediated by the reduction of COX-2, which was associated with inflammation triggered by ROS. Overall, the preclinical model could be used as a proof of concept to demonstrate the efficacy of anti-inflammatory combinatorial therapies by influencing oxidative stress. Similar research could provide a basis for more DNA-related cancer treatment research in the future.

• An inhibition of redox-responsive cyclooxygenase (COX) enzymes is often attributed to the mechanism of aspirin.• COX-2 regulates tumour growth, invasion and metastasis in breast cancer.

ROS and DNA damage
• Reactive oxygen species (ROS) are a group of short-lived, highly reactive, oxygencontaining molecules • induce DNA damage and affect the DNA damage response (DDR).
• COX-2 expression could be triggered by ROS • ROS favors the expression of an inflammatory phenotype that leads to the induction of COX-2.• CTCs are a rare subset of cells found in the blood of patients with solid tumors.
• We demonstrate an efficient approach to evaluate drug response using patient-derived CTC cultures obtained from liquid biopsy.Remove red blood cells prior to seeding

Significance of liquid biopsy and CTCs
• Cancer is a dynamic disease.During the course of disease, cancers generally become more heterogeneous.
• Heterogeneous response of therapy highlights the importance of patient-derived preclinical models.
• lack of a robust anticancer drug screening system to monitor patients during treatment.
• Our CTC assay obtained from liquid biopsy is efficient, non-invasive, inexpensive and drug screening for personalized treatment.

DA therapy reduced cancer stem cells
• Increase in CSCs post treatment have a high potential to form secondary metastases, leading to cancer relapse.
• CD44 + /CD24 − phenotype corresponded to the proportion of CSC-like cells • DA treatment with 500 mg/ml aspirin resulted in a significant reduction of CSC proportion.

DA therapy in clinical cohorts
• Representative images of microwell array with clusters (left) and without clusters (right).Scale bar is 50 µm.
• Patient samples that responded in terms of killing efficacy are marked in red.
• Heterogeneity of patient profiles and the reliable patient-derived models are importance for screening similar anti-inflammatory and anticancer strategies Khoo B L, Grenci G, Lim J S Y, et al. 2019, 120(4): 407.
• Calcein AM is also an indicator of intracellular oxidative activity.
• The differential intensity of Calcein AM suggests a reduction in intracellular oxidative activity in samples under both single drug aspirin and combinatorial DA therapy.
• Intracellular oxidative activity is a factor in metabolism and a key regulating process for several core functions including cell proliferation and transcription.• Compared with the treatment with doxorubicin alone, the intracellular oxidative activity in the sample under combinatorial DA treatment was reduced, as demonstrated by the intensity of Calcein AM.We demonstrated that the treatment outcomes were mediated by the reduction of COX-2, which was associated with inflammation triggered by ROS.
• Overall, the preclinical model could be used as a proof of concept to demonstrate the efficacy of antiinflammatory combinatorial therapies by influencing oxidative stress.Similar research could provide a basis for more DNA-related cancer treatment research in the future.