Tumor Volume Measurements Research Articles

Abstract P3-11-05: Efficacy of Gonadal steroids on aromatase inhibitor-resistant ER+ breast cancer: Insights from single-cell trajectory analysis of a patient-derived xenograft model

Abstract Background: While estrogen typically promotes the progression of hormone-dependent breast cancer via the activation of estrogen receptor (ER)-α, estrogen-induced suppression of ER+ breast cancer has been clinically observed. Our previous study demonstrated that estrogen increased the percentage of cells expressing IL24, linking to the estrogen-dependent growth inhibition of an ER+ aromatase inhibitor (AI)-resistant tumor (Mori et al. Cancers, 2021). To continue our evaluation, we investigated whether progesterone (P4) and dihydrotestosterone (DHT) would affect the growth of this estrogen-suppressive tumor. Methods: An estrogen-suppressive patient-derived xenograft (PDX) model (named GS3) was established from an AI resistant ER+/HER2– brain metastatic breast cancer. E2 (1mg), P4 (10mg), DHT (12.5mg), or placebo pellets were implanted in mice carrying GS3 for in vivo drug efficacy examination. Beside tumor growth response, immunohistochemistry (IHC) and RNA sequencing of PDX specimens were conducted to decipher molecular changes after each treatment. The single-cell RNAseq analysis was further performed to examine gene expression profiles in individual cells. Results: ERα, ERβ, Progesterone receptor (PR), and androgen receptor (AR) genes in GS3 are wild-type and are not amplified. Measurements of tumor volume showed that E2, E2+P4, and DHT suppressed the growth of GS3. The Tumor growth was not modulated by P4 treatment. IHC indicated that the number of Ki-67+ cells were decreased after E2, E2+P4, and DHT treatments, but were not changed after P4 treatment. PR+ cells appeared after E2 and E2+P4 treatments, and the AR+ cells increased after DHT treatment. Bulk-RNA sequencing indicated that E2 and E2+P4 treatments resulted in comparable gene expression patterns, while those of placebo and P4-treated tumors were similar. GSEA analysis showed that in E2 treatment, the hallmark estrogen response gene sets were upregulated, and the hallmark G2M checkpoint gene set was downregulated. However, in DHT treatment, the hallmark interferon alpha/gamma response gene sets and androgen response gene set were upregulated, and the hallmark TNFA signaling via NFkB gene set was downregulated. Single-cell RNA sequencing analysis of Placebo/E2/DHT samples revealed 9 clusters; cells from E2-treated and DHT-treated tumors were placed in different clusters based on principle component analysis of Highly Variable Genes, although both E2 and DHT treatments resulted in tumor regression. DHT promoted cell cycle arrest, but it did not increase IL24 expression. The hallmark oxidative phosphorylation and androgen response gene sets were upregulated in all clusters from DHT-treated tumors. Trajectory analysis of single cells revealed that three major branches associated with clusters selective to the treatments of placebo, E2, and DHT were separated from a common branch which are consist of G2M phase cells. Conclusions: E2 and DHT were effective suppressor of GS3, but not P4. Based on the results from IHC, bulk-RNAseq, and Single-cell RNAseq, the mechanism of DHT-induced tumor regression is different from that by E2. Our results suggest that DHT/E2 could be treatment options for patients with relapsed ER+ AI-resistance breast cancer. Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Jinhui Wang, Xiwei Wu, Noriko Kanaya, George Somlo, Shiuan Chen. Efficacy of Gonadal steroids on aromatase inhibitor-resistant ER+ breast cancer: Insights from single-cell trajectory analysis of a patient-derived xenograft model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-05.

Glial Cell Line-Derived Neurotrophic Factor Chitosan Nanolipid Carrier Inhibits Intestinal Carcinoma via Restraining NF-κB Activity

Background: Glial cell line derived neurotrophic factor (GDNF) is a potent neurotrophic factor, which has been shown to affect the metastasis and invasion of cancer cells. The molecular mechanism of GDNF is still unclear. Here, we investigate the mechanism of GDNF embedded in chitosan (CS) nano lipid carrier in colorectal cancer. Methods: Electron microscopy was used to detect the relationship between the characteristics of GDNF chitosan (CS) coated nano lipid carrier, MTT and apoptosis tests were used to detect the effect of GDNF chitosan nano lipid carrier expression on the proliferation ability of intestinal cancer cells, and immunofluorescence was used to detect the effect of GDNF chitosan nano lipid carrier expression on NF-KB signal. The results were verified by western-blot. In vivo using a mouse model. Results: GDNF chitosan nano lipid carrier showed positive zeta value, indicating that the process of CS coating GDNF was successful. OD values of nanocomposites at different concentrations were measured by UV spectrophotometer with MTT. Our data showed that the experimental group showed a relationship between concentration and dose dependent on tolerance growth, indicating that GDNF chitosan nano lipid carrier had better anti-tumor activity in colorectal cancer cell lines. Compared with most GDNF chitosan nano lipid carrier groups, the expression of NF-κB immunofluorescence detection signal was inhibited, and the expression of NF-κB was down-regulated in colorectal cancer. In vivo results showed that the measured tumor volume decreased after treatment with GDNF chitosan nanoparticle lipid carrier, and the survival of mice treated with GDNF chitosan nanoparticle lipid carrier was longer. Conclusion: GDNF chitosan nano lipid carrier can inhibit the expression of NF-κB signal and reduce the proliferation of tumor In vivo, thus slowing down the occurrence and development of intestinal cancer cells.

Predicting Patient-Specific Tumor Dynamics: How Many Measurements Are Necessary?

Acquiring sufficient data is imperative to accurately predict tumor growth dynamics and effectively treat patients. The aim of this study was to investigate the number of volume measurements necessary to predict breast tumor growth dynamics using the logistic growth model. The model was calibrated to tumor volume data from 18 untreated breast cancer patients using a varying number of measurements interpolated at clinically relevant timepoints with different levels of noise (0-20%). Error-to-model parameters and the data were compared to determine the sufficient number of measurements needed to accurately determine growth dynamics. We found that without noise, three tumor volume measurements are necessary and sufficient to estimate patient-specific model parameters. More measurements were required as the level of noise increased. Estimating the tumor growth dynamics was shown to depend on the tumor growth rate, clinical noise level, and acceptable error of the to-be-determined parameters. Understanding the relationship between these factors provides a metric by which clinicians can determine when sufficient data have been collected to confidently predict patient-specific tumor growth dynamics and recommend appropriate treatment options.

Downsizing of rectal cancer following neoadjuvant radiotherapy (5 × 5 Gy) and long interval surgery evaluated using MRI semiautomated volumetric measurements, a retrospective study.

Neoadjuvant conventional chemoradiation (CRT) is the standard treatment for primary locally non-curatively resectable rectal cancer, as tumor downsizing may allow R0 resectability. Short-term neoadjuvant radiotherapy (5x5 Gy) followed by an interval before surgery (SRT- delay) is an alternative for multimorbid patients who cannot tolerate CRT. This study examined the extent of tumor downsizing achieved with the SRT-delay approach in a limited cohort that underwent complete re-staging before surgery. Between March 2018 and July 2021, 26 patients with locally advanced primary adenocarcinoma (>uT3 or/and N+) of the rectum were treated with SRT-delay. 22 patients underwent initial staging and complete re-staging (CT, endoscopy, MRI). Tumor downsizing was assessed by staging and re-staging data and pathologic findings. Semiautomated measurement of tumor volume was performed using mint Lesion™ 1.8 software to evaluate tumor regression. The mean tumor diameter determined on sagittal T2 MRI images decreased significantly from 54.1 (23-78) mm at initial staging to 37.9 (18-65) mm at re-staging before surgery (p <0.001) and to 25.5 (7-58) mm at pathologic examination (p <0.001). This corresponds to a mean reduction in tumor diameter of 28.9 (4.3-60.7) % at re-staging and 51.1 (8.7-86.5) % at pathology. Mean tumor volume determined from transverse T2 MR images mint LesionTM 1.8 software significantly decreased from 27.5 (9.8 - 89.6) cm3 at initial staging to 13.1 (3.7 - 32.8) cm3 at re-staging (p <0.001), corresponding to a mean reduction of 50.8 (21.6 - 77) %. The frequency of positive circumferential resection margin (CRM) (less than 1mm) decreased from 45,5 % (10 patients) at initial staging to 18,2 % (4 patients) at re-staging. On pathologic examination, the CRM was negative in all cases. However, multivisceral resection for T4 tumors was required in 2 patients (9%). Tumor downstaging was noted in 15 of 22 patients after SRT-delay. In conclusion, the observed extent of downsizing is broadly comparable to the results of CRT, making SRT-delay a serious alternative for patients who cannot tolerate chemotherapy.

Therapeutic Performance Evaluation of 213Bi-Labelled Aminopeptidase N (APN/CD13)-Affine NGR-Motif ([213Bi]Bi-DOTAGA-cKNGRE) in Experimental Tumour Model: A Treasured Tailor for Oncology

Since NGR-tripeptides (asparagine-glycine-arginine) selectively target neoangiogenesis-associated Aminopeptidase N (APN/CD13) on cancer cells, we aimed to evaluate the in vivo tumour targeting capability of radiolabelled, NGR-containing, ANP/CD13-selective [213Bi]Bi-DOTAGA-cKNGRE in CD13pos. HT1080 fibrosarcoma-bearing severe combined immunodeficient CB17 mice. 10 ± 1 days after cancer cell inoculation, positron emission tomography (PET) was performed applying [68Ga]Ga-DOTAGA-cKNGRE for tumour verification. On the 7th, 8th, 10th and 12th days the treated group of tumourous mice were intraperitoneally administered with 4.68 ± 0.10 MBq [213Bi]Bi-DOTAGA-cKNGRE, while the untreated tumour-bearing animals received 150 μL saline solution. In addition to body weight (BW) and tumour volume measurements, ex vivo biodistribution studies were conducted 30 and 90 min postinjection (pi.). The following quantitative standardised uptake values (SUV) confirmed the detectability of the HT1080 tumours: SUVmean and SUVmax: 0.37 ± 0.09 and 0.86 ± 0.14, respectively. Although no significant difference (p ≤ 0.05) was encountered between the BW of the treated and untreated mice, their tumour volumes measured on the 9th, 10th and 12th days differed significantly (p ≤ 0.01). Relatively higher [213Bi]Bi-DOTAGA-cKNGRE accumulation of the HT1080 neoplasms (%ID/g: 0.80 ± 0.16) compared with the other organs at 90 min time point yields better tumour-to-background ratios. Therefore, the therapeutic application of APN/CD13-affine [213Bi]Bi-DOTAGA- cKNGRE seems to be promising in receptor-positive fibrosarcoma treatment.

Knockdown of PRKD2 Enhances Chemotherapy Sensitivity in Cervical Cancer via the TP53/CDKN1A Pathway.

Chemotherapy is the common treatment for cervical cancer, and the occurrence of drug resistance seriously affects the therapeutic effect of cervical cancer. Our previous study found that PRKD2 mutations occurred only in cervical cancer patients with chemotherapy resistance. However, the relationship between PRKD2 and drug resistance of cervical cancer remains unknown. We aim to clarify the relationship between PRKD2 and drug resistance of cervical cancer. Samples of patient tumor tissue were collected before chemotherapy and sequenced by WES. Chemotherapy clinical response was determined by measuring tumor volume. The expression of PRKD2, cell viability, and apoptosis were assessed by qRT-PCR, Western blot, CCK8, and flow cytometry in SiHa and ME180 cells after transfected with siPRKD2. The chemotherapy sensitivity signaling- related proteins were analyzed by Western blot. The expression levels of PRKD2 TP53, and CDKN1A in tissues were detected by immunohistochemistry staining. The expression of PRKD2 was higher in chemotherapy-resistant cervical cancer patients. PRKD2 knockdown increased the chemotherapy sensitivity of cervical cancer cells via the TP53/CDKN1A pathway, which led to G1 arrest and cell apoptosis. Furthermore, downregulation of PRKD2 enhances chemotherapeutic sensitivity in cervical cancer patients through the TP53/CDKN1A pathway. In summary, PRKD2 may be a promising therapeutic target to improve the efficacy of chemotherapy.

Mathematical Model of Triple-Negative Breast Cancer in Response to Combination Chemotherapies.

Triple-negative breast cancer (TNBC) is a heterogenous disease that is defined by its lack of targetable receptors, thus limiting treatment options and resulting in higher rates of metastasis and recurrence. Combination chemotherapy treatments, which inhibit tumor cell proliferation and regeneration, are a major component of standard-of-care treatment of TNBC. In this manuscript, we build a coupled ordinary differential equation model of TNBC with compartments that represent tumor proliferation, necrosis, apoptosis, and immune response to computationally describe the biological tumor affect to a combination of chemotherapies, doxorubicin (DRB) and paclitaxel (PTX). This model is parameterized using longitudinal [18F]-fluorothymidine positron emission tomography (FLT-PET) imaging data which allows for a noninvasive molecular imaging approach to quantify the tumor proliferation and tumor volume measurements for two murine models of TNBC. Animal models include a human cell line xenograft model, MDA-MB-231, and a syngeneic 4T1 mammary carcinoma model. The mathematical models are parameterized and the percent necrosis at the end time point is predicted and validated using histological hematoxylin and eosin (H&E) data. Global Sobol' sensitivity analysis is conducted to further understand the role each parameter plays in the model's goodness of fit to the data. In both the MDA-MB-231 and the 4T1 tumor models, the designed mathematical model can accurately describe both tumor volume changes and final necrosis volume. This can give insight into the ordering, dosing, and timing of DRB and PTX treatment. More importantly, this model can also give insight into future novel combinations of therapies and how the immune system plays a role in therapeutic response to TNBC, due to its calibration to two types of TNBC murine models.

Is there a role for tumor volume in prediction of prognosis for oral cancer?

PurposeNew prognostic factors in oral squamous cell carcinoma (OSCC) (tumor-, host-, and environment-related) have been introduced recently to complete those traditionally considered. Among them, tumor volume (TV) could be the most interesting and applicable in clinical practice, considering the routine use of computed tomography in tumor staging. In this retrospective study we aimed to investigate whether a correlation exists among these new prognostic factors and survival outcomes. Meterials and methodsWe collected data about 140 patients affected by OSCC who underwent primary surgery. Prognostic factors were collected and Overall Survival (OS), Disease Specific Survival (DSS) and Disease Free Survival (DFS) were estimated using Kaplan-Meier method; the Log-Rank test (Mantel-Cox) and Cox regression models were applied to investigate predictors of survival. ResultsThe 5-year OS, DSS and DFS were 73.6 %, 89.2 % and 75.2 % respectively. Nodal metastasis (pN+), relapse and American Society of Anesthesiologists ASA-II were found independent prognostic factors for OS, and significantly associated to worst DSS (p < 0.001). TV significantly correlated with higher relapse occurrence (p = 0.03). ConclusionsIn our experience, lymph-node status, ASA classification and relapse significantly influenced DSS on univariate analysis. TV could represent an interesting additional parameter, since it significantly influenced DFS. However, prospective studies with standardized TV measurements and a greater number of patients are needed to validate this result.

3D Ultrasound versus Computed Tomography for Tumor Volume Measurement Compared to Gross Pathology-A Pilot Study on an Animal Model.

The margin of the removed tumor in cancer surgery has an important influence on survival. Adjuvant treatments, prognostic complications, and financial costs are required when the pathologist observes a close/positive surgical margin. Ex vivo imaging of resected cancer tissue has been suggested for margin assessment, but traditional cross-sectional imaging is not optimal in a surgical setting. Instead, three-dimensional (3D) ultrasound is a portable, high-resolution, and low-cost method to use in the operation room. In this study, we aimed to investigate the accuracy of 3D ultrasound versus computed tomography (CT) to measure the tumor volume in an animal model compared to gross pathology assessment. The specimen was formalin fixated before systematic slicing. A slice-by-slice area measurement was performed to compare the accuracy of the 3D ultrasound and CT techniques. The tumor volume measured by pathological assessment was 980.2 mm3. The measured volume using CT was 890.4 ± 90 mm3, and the volume using 3D ultrasound was 924.2 ± 96 mm3. The correlation coefficient for CT was 0.91 and that for 3D ultrasound was 0.96. Three-dimensional ultrasound is a feasible and accurate modality to measure the tumor volume in an animal model. The accuracy of tumor delineation on CT depends on the soft tissue contrast.

Fibroblast Activation Protein-Targeted Radioligand Therapy for Treatment of Solid Tumors.

Fibroblast activation protein (FAP) has received increasing attention as an oncologic target because of its prominent expression in solid tumors but virtual absence from healthy tissues. Most radioligand therapies (RLTs) targeting FAP, however, suffer from inadequate tumor retention or clearance from healthy tissues. Herein we report a FAP-targeted RLT comprising an FAP6 ligand conjugated to DOTA and an albumin binder (4-p-iodophenylbutyric acid, or IP) for enhanced pharmacokinetics. We evaluated the performance of the resulting FAP6-IP-DOTA conjugate in 4 tumor models, 3 of which express FAP only on cancer-associated fibroblasts, that is, analogously to human tumors. Methods: Single-cell RNA-sequencing data were analyzed from 34 human breast, ovarian, colorectal, and lung cancers to quantify FAP-overexpressing cells. FAP6-DOTA conjugates were synthesized with or without an albumin binder (IP) and investigated for binding to human FAP-expressing cells. Accumulation of 111In- or 177Lu-labeled conjugates in KB, HT29, U87MG, and 4T1 murine tumors was also assessed by radioimaging or biodistribution analyses. Radiotherapeutic potency was quantitated by measuring tumor volumes versus time. Results: Approximately 5% of all cells in human tumors overexpressed FAP (cancer-associated fibroblasts comprised ∼77% of this FAP-positive subpopulation, whereas ∼2% were cancer cells). FAP6 conjugates bound to FAP-expressing cells with high affinity (dissociation constant, ∼1 nM). 177Lu-FAP6-IP-DOTA achieved an 88-fold higher tumor dose than 177Lu-FAP6-DOTA and improved all tumor-to-healthy-organ ratios. Single doses of 177Lu-FAP6-IP-DOTA suppressed tumor growth by about 45% in all tested tumor models without causing reproducible toxicities. Conclusion: We conclude that 177Lu-FAP6-IP-DOTA constitutes a promising candidate for FAP-targeted RLT of solid tumors.

MNG-3 STRATIFICATION OF TUMOR GROWTH BY RISK FACTORS AND PREDICTED TUMOR VOLUME CURVES FOR SUPRATENTORIAL MENINGIOMAS

Abstract Background The natural history of meningiomas is still unclear, and no guidelines have been established based on objective indices regarding the necessity and timing of therapeutic intervention.Objective: We attempted to provide statistics on the characteristics of tumor volume change, stratify tumor growth by risk factors, and generate a predictive tumor volume curve based on the statistics, with the aim of describing the natural history of meningiomas. Methods 313 cases were included in the study, with the origin of meningioma being the circumflex and parasagittal sinus areas and the cerebral sickle region, and with multiple MRI scans performed at intervals of at least 3 months. Relative growth rate (RGR) and annual volume change (AVC) were calculated by measuring tumor volume, and the patients were classified into three groups according to the combination of gender, age, and MRI T2WI signal intensity, and compared. Results The median RGR and AVC of the entire cohort were 6.1% and 0.20 cm3/year, respectively, and there were significant differences between groups in gender (p=0.018) and MRI T2WI (p &amp;lt; 0.001) for RGR and tumor location (p=0.025) and initial tumor volume (p &amp;lt; 0.001)for AVC. The median RGR and AVC in the classification were 17.5% and 1.05 cm3/year for the very high growth group, 8.2% and 0.33 cm3/year for the high growth group, and 3.4% and 0.04 cm3/year for the low growth group, showing significant differences between the groups (p &amp;lt; 0.001). The predicted tumor volume curve showed an average 2.24-fold or 5.24 cm3 increase in volume over 5 years in the very high-growth group, while little tumor volume change was observed in the low-growth group. Conclusion The combination of growth risk factors allowed stratification of tumor growth and provided a predictive tumor volume curve for each county. The results may assist in the treatment of meningiomas.

A retrospective diagnostic test study on circulating tumor cells and artificial intelligence imaging in patients with lung adenocarcinoma.

Either tumor volume or folate-receptor-positive circulating tumor cells (FR+CTC) has been proven effective in predicting tumor cell invasion. However, it has yet to be documented to use FR+CTC along with artificial intelligence (AI) tumor volume to differentiate between pathological subtypes of lung adenocarcinoma (LUAD). Therefore, this study is aimed to evaluate the accuracy of FR+CTC and AI tumor volume for classifying the invasiveness of LUAD. A total of 226 patients who were diagnosed with LUAD were enrolled. The inclusion criteria were: (I) FR+CTC detection and AI imaging before anticancer therapy, and (II) definite histopathologic diagnosis, which is the gold diagnosis of LUAD and its subtypes. Use the CytoploRare® Detection Kit to quantify FR+CTC and the AI-assisted diagnosis system, ScrynPro, to measure tumor volume. The clinical data were used to construct univariate and multivariate logistic regression models. A nomogram was drawn based on the multivariate logistic regression model. The validity is evaluated by the calibration curve and Hosmer-Lemeshow goodness-of-fit test. The mean age of 146 patients (96 males, 49 females and 1 gender missing) retrospectively enrolled was 56.6. In the cohort, 41 and 105 patients were assigned to adenocarcinoma in situ (AIS) + minimally invasive adenocarcinoma (MIA) and invasive pulmonary adenocarcinoma (IPA), respectively. There was no significant difference between the sex distribution and smoking history of the two groups (P=0.155 and P=0.442, respectively). In univariate analysis, the nodules type, maximum density, tumor volume and FR+CTC level were statistically significant with the invasiveness of LUAD (P<0.05). The multivariate analysis showed significant differences in FR+CTC and AI tumor volume (P<0.001). The area under the curves (AUCs) of FR+CTC and AI tumor volume in diagnosing tumor invasiveness were 0.659 and 0.698, respectively. A predictive model combining FR+CTC with AI tumor volume showed a sensitivity of 86.89% and a specificity of 70.94%, and the AUC was 0.841. The nomogram had good agreement with actual observation, and the Hosmer-Lemeshow test yielded non-significant goodness-of-fit. FR+CTC and/or AI tumor volume are independent indicators of the invasiveness of LUAD, and the nomogram based on them can be used for the preoperative screening of patients.

Folate receptor-targeted near-infrared photodynamic therapy for folate receptor-overexpressing tumors.

Photodynamic therapy (PDT) is a minimally invasive form of cancer therapy, and the development of a novel photosensitizer (PS) with optimal properties is important for enhancing PDT efficacy. Folate receptor (FR) membrane protein is frequently overexpressed in 40% of human cancer and a good candidate for tumor-specific targeting. Specific active targeting of PS to FR can be achieved by conjugation with the folate moiety. A folate-linked, near-infrared (NIR)-sensitive probe, folate-Si-rhodamine-1 (FolateSiR-1), was previously developed and is expected to be applicable to NIR-PDT. To investigate the therapeutic efficacy of NIR-PDT induced by FolateSiR-1, a FR-targeted PS, in preclinical cancer models. FolateSiR-1 was developed by conjugating a folate moiety to the Si-rhodamine derivative through a negatively charged tripeptide linker. FR expression in the designated cell lines was examined by western blotting (WB). The selective binding of FolateSiR-1 to FR was confirmed in FR overexpressing KB cells (FR+) and tumors by fluorescence microscopy and in vivo fluorescence imaging. Low FR expressing OVCAR-3 and A4 cell lines were used as negative controls (FR-). The NIR light (635 ± 3 nm)-induced phototoxic effect of FolateSiR-1 was evaluated by cell viability imaging assays. The time-dependent distribution of FolateSiR-1 and its specific accumulation in KB tumors was determined using in vivo longitudinal fluorescence imaging. The PDT effect of FolateSiR-1 was evaluated in KB tumor-bearing mice divided into four experimental groups: (1) FolateSiR-1 (100 μmol/L) alone; (2) FolateSiR-1 (100 μmol/L) followed by NIR irradiation (50 J/cm2); (3) NIR irradiation (50 J/cm2) alone; and (4) no treatment. Tumor volume measurement and immunohistochemical (IHC) and histological examinations of the tumors were performed to analyze the effect of PDT. High FR expression was observed in the KB cells by WB, but not in the OVCAR-3 and A4 cells. Substantial FR-specific binding of FolateSiR-1 was observed by in vitro and in vivo fluorescence imaging. Cell viability imaging assays showed that NIR-PDT induced cell death in KB cells. In vivo longitudinal fluorescence imaging showed rapid peak accumulation of FolateSiR-1 in the KB tumors 2 h after injection. In vivo PDT conducted at this time point caused tumor growth delay. The relative tumor volumes in the PDT group were significantly reduced compared to those in the other groups [5.81 ± 1.74 (NIR-PDT) vs 12.24 ± 2.48 (Folate-SiR-1), vs 11.84 ± 3.67 (IR), vs 12.98 ± 2.78 (Untreated), at Day 16, P < 0.05]. IHC analysis revealed reduced proliferation marker Ki-67-positive cells in the PDT treated tumors, and hematoxylin-eosin staining revealed features of necrotic- and apoptotic cell death. FolateSiR-1 has potential for use in PDT, and FR-targeted NIR-PDT may open a new effective strategy for the treatment of FR-overexpressing tumors.

CSIG-41. SENSITIZING CANCER CELLS TO TUMOR TREATING FIELDS (TTFIELDS) BY INHIBITION OF PI3K

Abstract Tumor Treating Fields (TTFields) are alternating electric fields, which disrupt cellular process critical for cancer cell survival and tumor progression. TTFields therapy is approved for the treatment of glioblastoma (GBM) and unresectable malignant pleural mesothelioma, and is being tested in clinical studies for the treatment of other solid tumors, including ovarian cancer, non-small cell lung carcinoma (NSCLC), and hepatocellular carcinoma (HCC). The current study aimed to detect potential mechanisms that may reduce cellular sensitivity to TTFields, and target these pathways in order to re-sensitize the cells to TTFields. Cancer cells (Ovarian A2780, GBM U-87 MG, and NSCLC H1299) that display reduced sensitivity to TTFields were generated by continuous long-term TTFields application (7 or 13 days, depending on the cell line). A Luminex multiplex assay revealed activation of the PI3K/AKT/mTOR signaling pathway in these cells, with significant increases in phosphorylation levels of AKT and RPS6. This elevation was also observed by immunohistochemistry in tumor sections from N1S1 HCC tumor-bearing rats treated with TTFields relative to sham. Treatment of cells with PI3K inhibitors re-sensitized them to TTFields and downregulated the phosphorylation of AKT. Concomitant application of TTFields with the PI3K inhibitor alpelisib in mice orthotopically implanted with MOSE-L firefly luciferase (FFL) ovarian cancer cells resulted in enhanced efficacy, as determined by In Vivo Imaging System (IVIS) measurements of tumor volume. Overall, this study demonstrated that the PI3K/AKT/mTOR signaling pathway is involved in reduced cancer cell sensitivity to long-term application of TTFields, and that re-sensitization may be achieved with relevant inhibitors. The results provide a rationale for further examining the potential benefit of TTFields concomitant with PI3K inhibitors.

Intravoxel Incoherent Motion MRI and PET in Locally Advanced Cervical Carcinoma Undergoing Concurrent Chemoradiation Therapy

<h3>Purpose/Objective(s)</h3> Dynamic contrast enhanced (DCE) CT or MRI and FDG PET have been used to evaluate the impact of tumor perfusion and metabolism on the course and effectiveness of concurrent chemoradiation therapy (CCRT) for cervical cancer. Combining perfusion and metabolic measures allows better identification of radioresistant sub-volumes: however, use of contrast and additional dose are barriers to frequent dual perfusion-metabolic imaging. Here, we investigated a contrast-free, non-ionizing alternative to DCE: intravoxel incoherent motion (IVIM) MRI which provides both perfusion and diffusion measurements. Our objective was to evaluate changes of PET and IVIM parameters in cervical cancer patients before and during CCRT and to correlate these parameters and their changes with tumor volume response during CCRT. <h3>Materials/Methods</h3> Pre- and on-treatment IVIM scans were performed on 15 cervical cancer patients undergoing definitive chemoradiation (median [range] age: 66 [29 to 85]; FIGO: 1 IB1, 1 IIA, 3 IIB, 1 IIIB, 1 IIIC, 5 IIIC1, 2 IIIC2, 1 IVA; hist: 11 squamous cell carcinoma, 3 endocervical adenocarcinoma, 1 large cell neuroendocrine carcinoma), of whom 14 also underwent pre-treatment PET scans. The on-treatment MRI occurred generally during the last week or shortly after completing EBRT. Diffusion weighted images (DWIs) were acquired at 3T scanners using a free-breathing single shot spin echo planar imaging (EPI) sequence. IVIM MRI maps were generated by Bayesian fitting of a two-compartment IVIM model to the DWIs. Three-dimensional co-registered maps of PET standardized uptake value (SUV) and IVIM diffusion coefficient (D), perfusion fraction (f), and pseudo-diffusion coefficient (D*) were generated. Summary statistics (mean +/- standard deviation) for these and other imaging parameters were calculated pre- and on-treatment. Univariate analysis was performed to determine relationships between relative change in gross tumor volume (ΔGTV) and pre-treatment imaging measurements. <h3>Results</h3> In tumors, pre- versus on-treatment values significantly changed for the following parameters: ΔGTV (66.8 +/- 43.2 vs. 32.0+/-23.0 ml, p<.001) and D (0.00109 +/- 0.00023 vs. 0.00120 +/- 0.00026 mm²/s, p=.02). The perfusion fraction f increased and D* decreased, but not significantly (f: 0.127 +/- 0.022 vs. 0.154 +/- 0.057 %, p=.07, D*: 0.0137 +/- 0.0035 vs. 0.0133 +/- 0.0039 mm²/s, p=.6). Pre-treatment SUV_max (r=.76, p=.002) and SUV_mean (r=.70, p=.007) were positively related with ΔGTV, while pre-treatment f, D*, and D were negatively but not significantly related with ΔGTV (r=-.44, p=.1; D*: r=-.14, p=.6; D: r=-.12, p=.7). <h3>Conclusion</h3> Both perfusion fraction and diffusion coefficient IVIM increased in locally advanced cervical carcinoma patients undergoing CCRT – reflecting changes in the tumor microenvironment during treatment. These weak relationships observed here suggest a potential complementary role and motivating the combined use of these modalities for tumor characterization.

Individualized Treatment Responsiveness as Quantified by Dynamic Changes in Cell Free HPV DNA in Patients Receiving Induction Chemotherapy and Definitive Chemoradiation for HPV-Related Head and Neck Cancer

<h3>Purpose/Objective(s)</h3> Studies have shown that induction chemotherapy (IC) is a viable pathway to chemoradiation therapy (CRT) de-intensification in HPV-related head and neck cancer (HNC). However, reliance on imaging alone for eligibility may incorrectly exclude cases where radiographic response lags or otherwise confounds biological response. We report serial cell free HPV DNA (cfHPV DNA) dynamics as a quantitative measure of early treatment responsiveness for HPV-related HNC patients receiving IC followed by CRT. <h3>Materials/Methods</h3> Starting Sept 26, 2021, we enrolled patients with locally advanced, high-risk HPV positive HNC who received 1-2 cycles of platinum/taxane IC followed by standard definitive CRT. Peripheral blood was collected biweekly during IC and weekly during CRT to measure cfHPV DNA levels using HPV-SEQ, a CLIA-certified assay that uses SafeSEQ technology to sensitively detect and quantify HPV16 and HPV18 DNA in plasma (Sysmex Inostics). The treating radiation oncologist delineated tumor volumes on pre- and post-IC CT simulation scans. <h3>Results</h3> At present, 86 plasma samples have been examined across 12 enrolled patients. The median age was 65 years (range: 35-79). The primary disease sites included 8 oropharynx (OPX), 2 nasopharynx, 1 sinonasal, and 1 larynx, with all tumors being high-risk HPV positive by in-situ hybridization. All patients had at least cT3 disease or cN3 disease by AJCC 8 criteria, with 7 patients having cT4 disease. Half of the patients have a >10 pack-year smoking history. Tumor volume measurements and cfHPV DNA levels at key time-points for the 8 patients who have completed IC are summarized in Table 1 below. <h3>Conclusion</h3> Serial cfHPV DNA may provide an earlier and more quantitative readout of individualized treatment responsiveness compared with imaging assessment alone in HPV-related HNC. We identify a group of patients with locally advanced disease who had complete or near-complete cfHPV DNA clearance during IC. Rapid and effective clearance of cfHPV DNA may ultimately serve as a preferred metric for identifying "exceptional responders" who are ideal biological candidates for short-course CRT after IC. Our granular cfHPV DNA dataset with biweekly measurements during IC and weekly measurements during CRT should lead to a more precise evaluation of cfHPV DNA clearance velocity and tumor response kinetics with this paradigm.

P2X7R promotes migration and invasion of Lewis lung cancer cells by activating the AKT signaling pathway

To explore the role of P2X7 receptor (P2X7R) in migration and invasion of mouse Lewis lung cancer (LLC) cells and examine the tumorigenic ability of LLC cells in P2X7R-knockout mice. RT-PCR was used to examine P2X7R mRNA expression in LLC cells. LLC cells were treated with ATP (as a P2X7R agonist) or 2'- 3'- O- (4-benzoyl- benzoyl)-ATP (BzATP) (a P2X7R agonist) with or without pretreatment with P2X7R antagonist oxATP or A438079. The changes in migration and invasive abilities of the cells were evaluated using wound healing assay and Transwell assay; Western blotting was performed to determine the activation level of the key proteins in the AKT signaling pathway. The effects of BzATP, A438079, and LY294002 (a inhibitor of the PI3K/AKT pathway) on migration and invasion of LLC cells were also examined. In wild-type (WT) and P2X7R knockout (P2X7-/-) C57BL/6 mice, the growth of subcutaneous LLC cell xenografts were observed by measuring tumor volume and weight. P2X7R expression was detected in LLC cells. Treatment with P2X7R agonist significantly enhanced migration and invasion abilities of LLC cells, and this effect was inhibited by application of P2X7R antagonists (P < 0.001). Western blotting showed that BzATP treatment of LLC cells significantly increased the expression level of p-AKT protein, which was obviously lowered by treatment with P2X7R antagonist (P < 0.01). P2X7R antagonist strongly inhibited BzATP-induced enhancement of LLC cell migration and invasion (P < 0.001). In the tumor- bearing mice, the tumor volume and weight were significantly lower in P2X7-/- mice than in WT mice (P < 0.05). P2X7R promotes migration and invasion of LLC cells by activating the AKT signaling pathway, and LLC cells show lowered tumorigenic capacity in P2X7-/- mice.

Evaluation of Antitumor and On-Target Activity of HDAC Inhibitors with the Zebrafish Embryo Xenograft Model.

Reliable preclinical drug testing models for cancer research are urgently needed with zebrafish embryo models emerging as a powerful vertebrate model for xenotransplantation studies. Here, we describe the evaluation of toxicity, efficacy, and on-target activity of histone deacetylase (HDAC) inhibitors in a zebrafish embryo yolk sac xenotransplantation model of medulloblastoma and neuroblastoma cells. For this, we performed toxicity assays with our zebrafish drug library consisting of 28 clinically relevant targeted as well as chemotherapeutic drugs with zebrafish embryos. We further engrafted zebrafish embryos with fluorescently labeled pediatric tumor cells (SK-N-BE(2)-C, HD-MB03, or MED8A) and monitored the progression after HDAC inhibitor treatment of xenotransplanted tumors through tumor volume measurements with high-content confocal microscopy in a multi-well format. The on-target activity of HDAC inhibitors was verified through immunohistochemistry staining on paraffin-embedded early larvae. Overall, the zebrafish embryo xenotransplantation model allows for fast and cost-efficient in vivo evaluation of targeted drug toxicity, efficacy, and on-target activity in the field of precision oncology.

Reproducibility of Three-Dimensional Volumetric Measurement of Periocular Tumor Models.

In this study, we measured the volume of customized tumor models in the periocular area using three-dimensional (3D) stereophotogrammetry and evaluated the reproducibility of these measurements. Five tumor models of different colors and sizes were placed in different periocular positions, and 3D facial images were obtained from 68 healthy adult volunteers. Subsequently, the volumes of the tumor models were measured, and the intra- and interrater reproducibility was assessed. The gray 6mm model revealed the highest reliable measurements in both Caucasians (intra- and interrater intraclass correlation coefficients of 0.981 and 0.899, mean absolute difference of 1.446 and 3.327mm3, relative error measurement of 3.497% and 8.120%, technical error of measurement of 1.450 and 3.105mm3, and relative technical error of measurement of 3.506% and 7.580%) and Asians (0.968 and 0.844, 1.974 and 4.067mm3, 4.772% and 9.526%, 2.100 and 4.302mm3, and 5.076% and 10.076%, respectively). The highest reliability of measurements in the lateral upper eyelid (0.88 and 0.95, 4.042 and 3.626mm3, 9.730% and 9.020%, 5.714 and 3.358mm3, and 9.730% and 8.350%, respectively) and medial upper eyelid (0.81 and 0.89, 4.313 and 4.226mm3, 9.730% and 9.020%, 6.098 and 4.069mm3, and 9.730% and 8.350%, respectively) with eyes closed was evident in Caucasians, while the same trend (0.841 and 0.815, 2.828 and 3.757mm3, 9.860% and 9.840%, 4.052 and 4.308mm3, and 9.860% and 9.740%, respectively) was observed in Asians in the medial canthus with eyes closed. This study confirms, for the first time, the high reliability of periocular tumor volume measurements using 3D stereophotogrammetry, suggesting its feasibility for eyelid tumor measurement. Further trials are required to investigate its clinical use for documentation and follow-up of different eyelid tumors.

Pancreatic Cancer-Derived Exosomes Promote the Proliferation, Invasion, and Metastasis of Pancreatic Cancer by the miR-3960/TFAP2A Axis.

Background The microRNAs (miRNAs) in cancer-derived exosomes have the ability to change tumor microenvironment. This study aims to investigate the role of miRNA in cancer-derived exosomes in pancreatic cancer (PC). Methods Based on the analysis of PC-derived and healthy exosomes by bioinformatics analysis and quantitative real-time PCR validation, the miR-3960 was identified to be the most significantly different miRNA, and TFAP2A proved as its potential target gene. Besides, the exosomes were isolated from PANC-1 cells and identified. After that, PANC-1 cells were treated with the isolated exosomes or transfected with miR-3960 mimics or si-TFAP2A, the effect of PC-derived exosomes, as well as the miR-3960/TFAP2A axis in PC cells, were assessed by the CCK-8, EDU staining, Transwell, cell colony formation, and flow cytometry assays. Furthermore, the effects of exosomes and the miR-3960/TFAP2A axis on PC tumor growth were observed in tumor-bearing mice by the measurement of tumor weight and volume, and hematoxylin-eosin staining. Moreover, the expressions of TFAP2A/PTEN/AKT signaling proteins were detected by Western blot. Results PC-derived exosomes were isolated successfully and proved to have promotion effects on the proliferation, metastasis, and invasion of PC cells both in vitro and tumor growth in vivo. Also, the PC-derived exosomes upregulated the TFAP2A, Bcl-2, and p-AKT/AKT protein levels, and inhibited PTEN and Bax levels and PANC-1 cell apoptosis. Overexpression of miR-3960 antagonized the promotion effect of exosomes on PC cells and the TFAP2A/PTEN/AKT signaling pathway, inhibiting the growth of tumors. Besides, si-TFAP2A enhanced the inhibitory effect of miR-3960 in PC. Conclusion MiR-3960 antagonizes the promotion effect of tumor-derived exosomes on the proliferation, invasion, and metastasis of PC via suppressing TFAP2A.