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Cancer patient tissueoid with self-homing nano-targeting of

metabolic inhibitor

1
2*
2*
2
1*
1
1
Hyo-Jin Yoon , Young Shin Chung , Yong Jae Lee , Seung Eun Yu , Sewoom Baek , Hye-Seon Kim , Sang Wun Kim ,
2†
Jung-Yun Lee , Sunghoon Kim , and Hak-Joon Sung 1†
2†

1 Department of Medical Engineering, Yonsei University College of Medicine, Seoul 03722, Republic of Korea.

2 Institute of Women's Life Medical Science, Department of Obstetrics and Gynecology, Yonsei University College of Medicine, Seoul 03722, Republic of Korea.

ABSTRACT INTRODUCTION

The current paradigm of cancer medicine focuses on patient- and/or cancer-specific Recent advances in biotechnologies have been integrated with precision medicine. As a

treatments, which has led to continuous progress in the development of patient major outcome, the paradigm of anti-cancer drug treatment has become to focus on

representatives (e.g., organoids) and cancer-targeting carriers for drug screening. As patient-specific selection and cancer-specific targeting [1, 2] . A common goal of current

breakthrough concepts, i) living cancer tissues convey intact profiles of patient-specific outstanding technologies is to expedite clinical success by cross-validating the safety,

microenvironmental signatures. ii) The growth mechanisms of cancer mass with intense efficacy, and efficiency of anti-cancer drugs among in vitro, in vivo, and clinical studies.

cell-cell interactions can be harnessed to develop self-homing nano-targeting by using To this end, extensive efforts have been made to develop artificial models (e.g., cell and

cancer cell-derived nanovesicles (CaNVs). Hence, we developed a tissueoid model of organoid) of patient representatives [3, 4] for drug screening as a potential platform for

ovarian cancer (OC) by culturing OC patient tissues in a 3D gel chip, whose cross-validation with in vivo responses through implantation [5, 6]. the breakthrough

microchannel networks enable perfusion to maintain the tissue viability. A novel model strategy of self-homing nano-targeting can be applied. The reciprocal interaction of cell

of systemic cancer responses was approached by xenografting OC tissueoids into membranes is a key mediator of cancer cell-cell interactions, whose function can be

ischaemic hindlimbs in nude mice. CaNVs were produced to carry general potentiated using cancer cell-derived nanovesicles (CaNV), owing to i) the same

chemotherapeutics or new drugs under pre/clinical studies that target the BRCA membrane characteristics as target cancer cells; ii) inanimation to prevent living cell-

mutation or energy metabolism, thereby increasing the test scope. This pioneer study mediated side effects; iii) effective systemic delivery similar to exosomes [7-9], as

cross-validates drug responses from the OC clinic, tissueoid, and animal model by revealed by another group [10]; and iv) more efficient mass production and purification

demonstrating the alignment of results in drug type-specific efficiency, BRCA mutation- [11, 12] compared to exosomes [13-16]. Moreover, the loading of anti-cancer drugs into

dependent drug efficiency, and metabolism inhibition-based anti-cancer effects. Hence, CaNV represents a “Trojan horse” strategy to inhibit cancer action synergistically with the

this study provides a directional foundation to accelerate the discovery of patient- self-homing nano-targeting approach. Therefore, this study introduces the novel concept

specific drugs with CaNV application towards future precision medicine. of an implantable OC tissueoid capable of culturing OC patient tissues in vitro and in vivo.

Drug type-specific efficiency, BRCA mutation-dependent drug efficiency, and metabolism

inhibition-based anti-cancer effects were tested. CaNVs were produced to deliver general
OBJECTIVES chemotherapeutics, as well as the two aforementioned drugs following the concept of

Trojan horse. Based on more than 100 patient records, the cross-validation among in

To demonstrate breakthrough concepts including the OC tissueoid, CaNV, new drugs, in vivo vitro, in vivo, and clinical studies were carried out as the first step of a paradigm shift in

model of systemic cancer response, and to cross-check with clinical outcomes. the field of new drug development.

RESULTS

Figure 3. Xenografting OC tissueoid as a new systemic model to cross-
Figure 2. Cancer cell derived-nanovesicles (CaNVs) for self-homing nano- validate in vivo anti-cancer effects of MB-Drug. (a) At week seven post OC

targeting of MB-Drug. (a) CaNV is produced by serial filtering of OC cells with tissueoid xenografting into ischemic hindlimbs of nude mice, tumor growth over 200

decreasing pore sizes and loading with the MB-Drug (i.e., a combination of mm in volume was established. Then, test groups (no treatment– PBS vehicle
3
gossypol and phenformin) by electroporation. CaNVs+/–MB-Drug are control, CaNVs, CaNVs+MB-Drug, MB-Drug) were injected intraperitoneally every

characterized for (b) size by DLS and (c) morphologies by TEM. (d) The loading week until sacrifice. Tumor tissues were harvested at week 14. Anti-cancer effects

of MB-Drug into CaNV was verified by analysing each component with MALDI- of test groups were determined in terms of (b) optical size imaging with a ruler, (c)

TOF/TOF. (e) Cancer-specific cytotoxic effects of test groups were determined by tumor weight (gram: g), and (d) quantitative analyses of progressive changes in

analysing the viability of normal vs. OC tissueoid with confocal image analysis, tumor size (mm ). (e) The mitotic index, proliferation (Ki67), and Notch-1 factor
3
+
which is further supported by (f) OC cell (PAX8 )-specific targeting and uptake of expression (cleaved Notch-1 and HES-1) were analysed quantitatively with
CaNV+MB-Drug in mixture culture with normal ovarian cells by phase contrast immune-histological staining (arrows in H&E images: apoptotic cells), (f) followed by

and immunofluorescence imaging. Data = mean ± S.E.M (n  3). *p < 0.05, **p Western blot analyses of Notch signaling proteins for crosschecking. Data = mean

< 0.01, and ***p < 0.001 versus no treatment or between lined groups. ± S.E.M (n = 6 mice). *p < 0.05, **p < 0.01, and ***p < 0.001 versus No treat or

between lined groups.

Figure 1. Clinically matched drug responses of ovarian cancer (OC) tissueoid

with introduction of anti-cancer metabolic (MB)-Drug. (a) The tissueoid is

defined as a culture of patient tissues in a 3D chip under perfusion of media (+/–

drug) into microchannel (vascular) networks at a flow rate of 20 μL/min. (b) OC

marker expression (PAX8 and p53) and histology of tissueoids were matched with

those of tissue samples from high-grade serious OC patients in-clinic, analysed by

optical examination with immunohistology and H&E staining. (c) The gelatin gel

allows the progressive cell invasion from OC patient tissues during 28-day culture

post embedding into the gel, as observed by optical imaging. (d) The viability of the

OC tissueoid was maintained during 30 days of perfusion culturing, as determined

by confocal imaging. (e) Gossypol and phenformin were used in combination as an

anti-cancer energy metabolism (MB)-Drug. (f) To represent patient tissue [+/- BRCA

mutation (mut)], drug type-dependent viabilities of OC tissueoids were validated

under treatments with paclitaxel (PTX) + carboplatin (CBP), olaparib (Ola), or MB-

Drug by confocal imaging analysis. Data = mean ± S.E.M (n 3). *p < 0.05, **p

< 0.01, and ***p < 0.001 versus no treatment or between lined groups.

Figure 4. Aligned drug responses between OC tissueoid and patient tissue in-

clinic. (a) During clinical progress in OC treatment, tissues were obtained from

diagnosis (*) and surgery in-between PTX+CBP (**) treatment for the drug-first

group (top) or surgery before the first PTX+CBP (***) administration for the surgery-

first group (bottom). (b) In alignment with the tissuoid responses, the first PTX+CBP

therapy exhibits anti-cancer effects (>50 %) on BRCA-WT and BRCA-mut groups (n

= 82). However, significant recurrence rates (>30 %) indicate the need to use

another drug. (c) Hence, Ola was administered to each group as a maintenance

therapy after PTX+CBP, resulting in a more effective tumor reduction (14.3 %) in the

BRCA-mut group compared to the BRCA-WT group (29.4 %). (d) As an example of

alignment with the OC tissueoid response, a clinical case is shown post first

PTX+CBP therapy. The effective drug response is evidenced by the significantly

lowered level of the standard tumor marker (CA 125: 13.7 U/mL) below the normal

limit (35 U/mL), which was verified by PET/CT imaging. (Red circle: tumor mass).

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