Repurposing Drugs Guided by Tissue Slice Screening in Cancer Patients Without Standard Treatment
Conditions: Cancer Patients With Solid Tumors, Cancer (Advanced Stage)
Sponsor: China Medical University Hospital

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Clinical Study Protocol 
Cover Page 
Document Type: Study Protocol  
Official Title: Repurposing drugs guided by tissue slice screening in cancer 
patients without standard treatment 
ClinicalTrials.gov Identifier (NCT Number): Not yet assigned  
Document Date: 4 July 2025  
Document Version: Version 1.0  
Principal Investigator: Ching-Yun Hsieh, MD  
Institution: China Medical University Hospital, Taichung, Taiwan 

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Abstract: 
Precision oncology demands robust methodologies to guide the selection of effective, 
personalized therapies. We developed OPTin (Organotypic Platform for Therapeutic 
Investigation), a tissue slice–based functional drug screening platform that integrates 
ex vivo tissue slicing, optical clearing, and high-resolution confocal imaging to 
evaluate drug-induced apoptosis at spatial resolution. In a patient with advanced 
hypopharyngeal carcinoma, OPTin screening identified venetoclax as an effective 
agent against the primary tumor, although the metastatic lesion remained resistant. A 
second OPTin analysis using metastatic tissue identified lenvatinib as a sensitizing 
agent. The combination therapy of venetoclax and lenvatinib led to a near-complete 
clinical remission and a marked reduction in serum AFP levels from 1077 to 371 
ng/mL. These findings highlight the potential of OPTin to provide spatially resolved, 
functional assessment of drug responses, enabling tailored treatment decisions in 
complex oncologic scenarios. Based on this proof-of-concept, we aim to systematically 
evaluate OPTin in a prospective clinical trial across diverse cancer types. 
Key Words: 
Tissue slice culture, drug screening, cell apoptosis. 

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Introduction: 
Precision medicine is transforming cancer treatment by replacing uniform protocols with 
therapies tailored to the genetic, molecular, and environmental profiles of individual patients 
and their tumors.1,2 Tumor heterogeneity arises from genetically diverse subclones that 
coexist and evolve within a single tumor, contributing to varied therapeutic responses.3 To 
address this complexity, precision tools such as multi-region sequencing and biomarker 
testing reveal actionable mutations and predictive markers, enabling molecularly guided 
therapies with improved efficacy over conventional chemotherapy.4 By aligning treatment 
strategies with tumor-specific biology, precision medicine intends to enhance survival 
outcomes, reduces treatment-related burden, and offers the potential for durable disease 
control. 
Three-dimensional culture systems better replicate the in vivo tumor microenvironment than 
traditional 2D models by preserving cell–cell and cell–matrix interactions, biochemical 
gradients, and tissue-specific barriers to drug delivery.5 Among these, organotypic tissue slice 
cultures offer a physiologically relevant, cost-effective platform that retains native 
extracellular matrix and immune components.6 Originally used to evaluate chemotherapeutic 
efficacy7, this approach has been extended to immune checkpoint8 and CAR-T9 therapies. 
While the approach holds promise for ex vivo drug testing, its clinical application in guiding 
patient-specific treatment decisions remains limited due to current technological constraints 
in drug screening and reproducibility.6 In particular, the conventional analytical 
method−relying on slicing tissue sections into 3–5 μm thin sections for immunohistochemical 
(IHC) analysis−may introduce sampling bias, contributing to poor reproducibility across 
experiments. 
To address the need for physiologically relevant, patient-specific drug testing, we developed 
OPTin (Organotypic Platform for Therapeutic Investigation), a screening system that 
integrates organotypic tissue slice cultures, optical clearing, and high-resolution fluorescence 
imaging. This platform enables spatially resolved assessment of drug-induced apoptosis 
within preserved tumor microenvironments and supports rapid evaluation of multiple agents 
for screening, including combination therapies. In our previous study, OPTin was applied to 
head and neck cancer specimens to assess the efficacy of chemotherapeutics, targeted drugs, 
and immune checkpoint inhibitors, with the goal of guiding compassionate treatment in 
late-stage disease. 
Methods: 
Clinical background of the patient 

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A 54-year-old male presented with persistent hoarseness and was eventually diagnosed with 
poorly differentiated carcinoma of the hypopharynx, exhibiting yolk sac tumor features. He 
initially deferred standard treatment and re-presented two months later with acute airway 
obstruction, necessitating emergency endotracheal intubation followed by tracheostomy. He 
received the bleomycin/etoposide/cisplatin (BEP) chemotherapy from November 2023 to 
February 2024, resulting in a partial response with decreased serum AFP levels. However, 
disease progression ensued, marked by rising AFP levels and new cervical lymph node 
metastases with extranodal extension. Re-biopsy of the cervical lymph node revealed a 
carcinoma with concurrent germ-cell-like and squamous differentiations. A second-line 
regimen of gemcitabine and oxaliplatin (GemOx), followed by pembrolizumab plus 
cisplatin/5-FU (PF), failed to control disease progression. Due to progressive airway 
compromise, a second tracheostomy was performed in December 2024, followed by 
concurrent chemoradiotherapy administered from December 2024 to January 2025. The 
patient's therapeutic timeline, including the administration of treatment regimens, is depicted 
in Figure 1A. 
 
Figure 1. Clinical course and histological and immunohistochemical analysis. 
(A) Timeline summarizing the treatment history from November 2023 to April 2025. 
OPTin analysis 
The tissue specimen (approximately (0.5 cm)3) was sectioned into 500 µm slices with a tissue 
matrix within 2 hours after surgical removal from China Medical University Hospital. Tissue 
slices were placed on SPONGOSTAN™ sponges (Ethicon, #MS0005) and cultured in RPMI 
medium (Gibco, #31800105) supplemented with 20% fetal bovine serum (Corning, 
#35-010-CV) and antibiotic–antimycotic solution (Corning, #30-004-CI) in a humidified 
incubator at 37°C with 5% CO₂. A total of 15 agents, including single and combination 
therapies, were evaluated in this study. The selection of investigational drugs was guided by 
institutional approval and constrained by the patient’s financial accessibility. Each drug was 
administered at a concentration of 10 μM10, and tissue slices were incubated for two days to 
allow drug exposure. Following this, tissue slices underwent fixation for one day, 
permeabilization, and subsequent immunostaining, which spanned a total of ten days. 
Apoptosis was visualized using cleaved caspase-3 antibody staining, while cell nuclei were 
identified by DAPI staining. In groups treated with immune checkpoint inhibitors, additional 

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staining for IFN-γ was performed to assess immune cell activation. After immunostaining, the 
samples were then processed using SeeDB tissue clearing protocol11, a technique that 
enhances optical transparency for deeper imaging, over a five-day period. Cleared tissues 
were imaged using Dragonfly confocal microscopy with optical sections acquired at 5-μm 
intervals. For tissue slices measuring 500 μm in thickness, a total of 100 sequential layers 
were scanned and digitally reconstructed into a composite three-dimensional image, enabling 
detailed visualization and analysis of drug responses at both the cellular and subcellular levels. 
Thus, this procedure takes total 18 days to identify which drugs might be positive. 
Preliminary Results: 
Histopathological and immunohistochemical analysis 
Despite multiple lines of treatment, the therapeutic response of the patient with the head and 
neck cancer remained limited (Fig. 1A and Method), prompting a re-evaluation of the tumor's 
pathological and molecular features through discussion in a multidisciplinary molecular 
tumor board. Histopathological analysis of the primary and metastatic tumor tissues revealed 
a squamous cell carcinoma with germ cell-like differentiation (Fig. 1B). Immunohisto- 
chemically, sal-like protein 4 (SALL4) and alpha-fetoprotein (AFP) are germ cell markers, 
cytokeratin 7 (CK7) is a non-keratinizing epithelial marker and p40 is a squamous 
differentiated marker. The initial biopsy of primary hypopharyngeal tumor revealed sheeting 
basophilic, round tumor cells exhibiting co-expression of CK7 and SALL4, but lack of p40 and 
AFP expression in the solid and glandular (yolk sac–like) patterns (Fig. 1C), suggestive of germ 
cell–like differentiation. Notably, a repeat biopsy of the primary hypopharyngeal tumor 
demonstrated strong AFP expression in the germ cell–like tumor cells, supporting germ 
cell-like tumor cells with a yolk sac differentiation (Fig. 1E-G). The metastatic carcinoma 
within the cervical lymph node shared partial histological similarity to the primary lesion, 
including the presence of germ cell–like differentiated components. However, regions of 
well-differentiated squamous cell carcinoma with prominent keratinization were also 
identified (Fig. 1D). Immunohistochemical stain demonstrated diffuse p40 positivity in the 
squamous components, while germ cell–like regions remained p40-negative, indicating 
divergent differentiations within the same lesion (Fig. 1H-I). 

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Figure 1. Clinical course and histological and immunohistochemical analysis. 
(B and C) Representative H&E-stained sections demonstrating the primary hypopharyngeal 
carcinoma exhibiting round to polygonal tumor cells with germ cell-like differentiation [T] and 
yolk sac features (arrow) in a vascular-rich [V] stroma. (D) H&E-stained sections 
demonstrating a metastatic carcinoma in a lymph node with coexisting germ cell-like 
differentiation as the same as primary lesion, and squamous differentiation with keratin 
formation [★]. B to D, original magnification, 200x. (E to I) Immunohistochemically, germ 
cell-like tumor cells showing positive reactivity to CK7 (E), SALL4 (F) and AFP (G), confirming 
the germ cell-like nature. The p40 showing a diffuse nuclear positivity in squamous 
differentiated components (H) and a negative staining in germ cell-like components (I). Brown 
color as a positive staining. E to I, original magnification, 100x. 
Whole exome sequencing (WES) analysis 
To identify potential therapeutic targets, WES12 was performed on both the primary and 
metastatic tumors of the patient with poorly differentiated hypopharyngeal adenocarcinoma 

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exhibiting yolk sac features. Shared mutations were identified in TP53, NOTCH1, FAT1, and 
CSF1R, while the metastatic lesion also harbored PIK3CA and NFE2L2 alterations. These 
findings suggest potential avenues for targeted therapy, including PI3K/AKT/mTOR and 
CSF1R-mediated pathways. While PIK3CA (p.E542K) is a well-characterized activating 
mutation and may confer sensitivity to PI3K inhibitors such as alpelisib, its role in head and 
neck cancer has only recently been evaluated in clinical trials.13 However, similar to the CSF1R 
mutation (p.V784M), which was detected at a low variant allele frequency, the PIK3CA 
mutation may represent a passenger alteration rather than a true driver event, thereby 
limiting its therapeutic relevance. Together, the co-occurrence of multiple mutations and the 
genomic divergence between primary and metastatic sites highlight the complexity of this 
tumor and the limitations of genomics alone in guiding treatment selection, underscoring the 
need for integrated functional platforms in precision oncology. 
OPTin analysis 
To address these therapeutic challenges, we developed OPTin (Fig. 2A), a functional drug 
screening platform that integrates ex vivo organotypic tissue slice culture with tissue clearing 
and high-resolution confocal imaging to assess patient-specific drug responses within a 
physiologically relevant microenvironment. Application of OPTin to the patient’s primary 
hypopharyngeal tumor tissue revealed the most extensive apoptosis following venetoclax 
treatment, indicating its potential as an effective therapeutic agent (Fig. 2B). The average 
fluorescence intensity (aFI) of cleaved caspase-3, a marker of apoptosis, was displayed in the 
lower left corner of each fluorescence image. Notably, the administration of venetoclax as a 
compassionate therapy (IRB identifier: CMUH110-REC2-097 by the Ethics Committee of China 
Medical University Hospital, Taichung, Taiwan) in this case led to complete regression of the 
hypopharyngeal tumor (Fig. 2C). 
Although venetoclax demonstrated significant inhibitory activity against the primary 
hypopharyngeal tumor, it showed no apparent efficacy in the metastatic cervical lymph node 
tissue, which exhibited no signs of regression (Data not shown). To further investigate this 
discrepancy, we re-evaluated the apoptotic effects of the previously tested drugs on the 
metastatic lesion. Confocal fluorescence imaging revealed a strikingly differential drug 
response between the primary and metastatic tumors (Fig. 2B vs. 3A), highlighting the 
presence of intratumor heterogeneity, consistent to the WES analysis. In line with the 
observed lack of clinical response, the metastatic tumor showed resistance to venetoclax 
despite its efficacy in the primary site. 

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Figure 2. Drug-induced apoptosis profiling in primary hypopharyngeal carcinoma 
tissues using the OPTin platform. 
(A) Schematic overview of the OPTin platform for ex vivo drug screening. The workflow 
integrates freshly resected tumor tissue slicing, drug treatment on sponge-supported culture, 
tissue clearing, immunofluorescence staining, and high-resolution confocal imaging to 
evaluate 
patient-specific 
therapeutic 
responses 
in 
a 
physiologically 
relevant 

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microenvironment. (B) Ex vivo tissue slices from the primary hypopharyngeal tumor were 
treated with the indicated therapeutic agents. Fluorescence imaging was performed using 
Dragonfly confocal microscopy. Green indicates cleaved caspase-3; DAPI stains nuclear DNA. 
The average fluorescence intensity (aFI; integrated density/area) was analyzed using ImageJ 
1.54g software. Scale bars, 200 μm. (C) Representative fiberoptic nasopharyngolaryngoscopy 
images obtained before and after venetoclax treatment, showing complete regression of the 
hypopharyngeal tumor. Red arrow, tumor. 
Among the agents tested, lenvatinib demonstrated the greatest apoptotic effect on the 
metastatic tumor tissue (Fig. 3A). Based on this finding, we initiated a second compassionate 
therapy combining lenvatinib with venetoclax. Following the combination treatment, the 
cervical lymph node metastasis decreased in size even 3 weeks post treatment. Computed 
tomography (CT) analysis revealed partial regression of the metastatic lesion, with residual 
soft tissue density still present in the left cervical region post-treatment (Fig. 3B). 
Concurrently, serum AFP levels declined significantly from 1077 to 371 ng/mL (Fig. 3C). These 
results suggest that the organotypic tissue slice drug screening platform may effectively guide 
drug selection in the context of complex tumor heterogeneity, offering a promising strategy to 
achieve personalized and therapeutically meaningful outcomes. 
 

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Figure 3. Drug-induced apoptosis profiling in metastatic lymph node carcinoma tissues 
using the OPTin platform. 
(A) Ex vivo tissue slices from the metastatic lymph node tumor were treated with the 
indicated therapeutic agents. Fluorescence imaging was performed using Dragonfly confocal 
microscopy. Green indicates cleaved caspase-3; DAPI stains nuclear DNA. The average 
fluorescence intensity (aFI; integrated density/area) was analyzed using ImageJ 1.54g 
software. Scale bars, 200 μm. (B) Axial and coronal CT images obtained before and after 
combined venetoclax and lenvatinib treatment. In December 2024, both the primary tumor 
(red arrow) and metastatic cervical lymph node lesions (blue arrow) were visible. By April 
2025, the primary tumor had regressed, consistent with the findings shown in Fig. 2C; 
however, residual metastatic tumor (indicated by the blue dotted arrow) had decreased in size 
but remained detectable. Yellow line, the largest dimension of tumors. (C) Timeline of clinical 
interventions, monitoring, and therapeutic strategies. The key procedures include WES, 
surgery, the OPTin assay, histopathological evaluation, imaging studies, and endoscopic 
assessment. 
Study Design 
This study aims to enroll 200 subjects from whom fresh tumor tissue specimens will be 
collected for ex vivo analysis. The OPTin platform will be used to evaluate the efficacy of 
FDA-approved anticancer drugs by screening their ability to induce apoptosis in tumor 
tissues across multiple cancer types. Based on the results of the OPTin assay, potential 

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therapeutic drug options will be recommended for patients who no longer have standard 
treatment options available. 
If the treating physician elects to adopt the drug recommendation generated by the OPTin 
platform, imaging-based clinical evaluations of the subject will be collected at each scheduled 
assessment. The therapeutic response to the selected treatment will be monitored for up to 
one year following initiation of therapy. 
Study Procedures and Laboratory Analyses 
Fresh tumor tissue specimens obtained during surgery must be larger than 0.5 cm³. 
Immediately after surgical collection, the tissue will be sectioned into 500 μm slices using a 
vibratome. Tissue slices will then be cultured and exposed to candidate drugs for 48 hours. 
Following drug treatment, the tissue slices will undergo immunofluorescence staining and 
tissue clearing procedures. High-resolution confocal imaging will subsequently be 
performed to quantify drug-induced apoptosis and evaluate features of the tumor 
microenvironment. The results of these analyses will be used to generate drug sensitivity 
profiles to assist clinicians in selecting potential therapeutic agents. 
Inclusion Criteria 
Subjects must meet all of the following criteria to be eligible for participation in the study: 
1. Adults ≥ 18 years of age. 
2. Willing and able to provide written informed consent prior to participation in any 
study-related procedures. 
3. Willing to undergo surgical collection of tumor tissue during the screening period, with a 
specimen size greater than 0.5 cm³. 
4. Willing to allow the use of their previous clinical test results and residual tissue 
samples for study-related analyses. 
Exclusion Criteria 
Subjects meeting any of the following criteria will be excluded from the study: 
1. 
Inability to obtain sufficient tumor tissue through surgical procedures. 
2. 
The treating physician decides not to adopt the drug recommendation generated by the 
study platform. 
3. 
The principal investigator determines that the subject is not suitable for enrollment in the 
study. 

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References: 
1. Haendel MA, Chute CG, Robinson PN. Classification, ontology, and precision medicine. N 
Engl J Med 2018;379:1452-62. 
2. Johnson DE, Burtness B, Leemans CR, et al. Head and neck squamous cell carcinoma. Nat 
Rev Dis Primers 2020;6:92. 
3. Parikh AS, Yu VX, Flashner S, et al. Patient-derived three-dimensional culture techniques 
model tumor heterogeneity in head and neck cancer. Oral Oncol 2023;138:106330. 
4. Duan XP, Qin BD, Jiao XD, et al. New clinical trial design in precision medicine: discovery, 
development and direction. Signal Transduct Target Ther 2024;9:57. 
5. Aksoy SA, Earl J, Grahovac J, et al. Organoids, tissue slices and organotypic cultures: 
advancing our understanding of pancreatic ductal adenocarcinoma through in vitro and ex 
vivo models. Semin Cancer Biol 2025;109:10-24. 
6. He L, Deng C. Recent advances in organotypic tissue slice cultures for anticancer drug 
development. Int J Biol Sci 2022;18:5885-96. 
7. Willoughby HW, Maughan GB, Tremblay PC, et al. Determination of individual human 
tumor sensitivity to antitumor agents by tissue-slice incubation. Can J Surg 1971;14:406-9. 
8. Sivakumar R, Chan M, Shin JS, et al. Organotypic tumor slice cultures provide a versatile 
platform for immuno-oncology and drug discovery. Oncoimmunology 2019;8:e1670019. 
9. Sullivan KM, Jiang X, Guha P, et al. Blockade of interleukin 10 potentiates antitumor 
immune function in human colorectal cancer liver metastases. Gut 2023;72:325-37. 
10. Broutier L, Mastrogiovanni G, Verstegen MM, et al. Human primary liver cancer-derived 
organoid cultures for disease modeling and drug screening. Nat Med 2017;23:1424-35. 
11. Ke MT, Fujimoto S, Imai T. SeeDB: a simple and morphology-preserving optical clearing 
agent for neuronal circuit reconstruction. Nat Neurosci 2013;16:1154-61. 
12. Solis RN, Silverman DA, Birkeland AC. Current trends in precision medicine and 
next-generation sequencing in head and neck cancer. Curr Treat Options Oncol. 
2022;23:254-67. 
13. Keam B, Hong MH, Shin SH, et al. Personalized biomarker-based umbrella trial for patients 
with recurrent or metastatic head and neck squamous cell carcinoma: KCSG HN 15-16 
TRIUMPH Trial. J Clin Oncol 2024;42:507-17. 
14. DiNardo CD, Pratz KW, Letai A, et al. Safety and preliminary efficacy of venetoclax with 
decitabine or azacitidine in elderly patients with previously untreated acute myeloid 
leukemia: a non-randomized, open-label, phase 1b study. Lancet Oncol 2018;19:216-28. 
15. Hoetelmans R, van Slooten HJ, Keijzer R, et al. Bcl-2 and Bax proteins are present in 
interphase nuclei of mammalian cells. Cell Death Differ 2000;7:384-92. 
16. Xie C, Edwards H, Caldwell JT, et al. Obatoclax potentiates the cytotoxic effect of cytarabine 
on acute myeloid leukemia cells by enhancing DNA damage. Mol Oncol 2015;9:409-21. 
17. Misra S, Moro CF, Del Chiaro M, et al. Ex vivo organotypic culture system of precision-cut 

A clinical trial of OPTin system for drug screening 20250704 Version 1 
Page 13 of 14 
  Protocol Version: 1.0 
  Version Data: July 4, 2025 
slices of human pancreatic ductal adenocarcinoma. Sci Rep 2019;9:2133. 
18. Lim CY, Chang JH, Lee WS, et al. Organotypic slice cultures of pancreatic ductal 
adenocarcinoma preserve the tumor microenvironment and provide a platform for drug 
response. Pancreatology 2018;18:913-27. 
19. Centenera MM, Hickey TE, Jindal S, et al. A patient-derived explant (PDE) model of 
hormone-dependent cancer. Mol Oncol 2018;12:1608-22. 
20. Xing F, Liu YC, Huang S, et al. Accelerating precision anti-cancer therapy by time-lapse and 
label-free 3D tumor slice culture platform. Theranostics 2021;11:9415-30. 

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